Antigen-binding molecule inducing immune response to target antigen

Abstract

The present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes depending on ion concentration conditions and containing an FcRn-binding domain having FcRn-binding activity in a neutral pH range, immune responses to the antigen are induced. Furthermore, the present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes depending on ion concentration conditions and containing an FcRn-binding domain having FcRn-binding activity in a neutral pH range, immune responses to the antigen are induced, and also the antigen-binding molecule has cytotoxicity or antiproliferative action against cancer cells, foreign biological species, or such that express the antigen to which the antigen-binding molecule binds.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 14 / 347,448, filed on Mar. 26, 2014 (now U.S. Pat. No. 10,556,949), which is the National Stage of International Application Serial No. PCT / JP2012 / 075043, filed on Sep. 28, 2012, which claims the benefit of Japanese Application Serial No. 2011-216958, filed on Sep. 30, 2011.TECHNICAL FIELD

[0002] The present invention provides pharmaceutical compositions comprising as an active ingredient an antigen-binding molecule that induces an immune response to a target antigen, or therapeutic methods that use the pharmaceutical compositions. The present invention also provides pharmaceutical compositions comprising as an active ingredient, an antigen-binding molecule that induces the above-mentioned immune response and also has cytotoxicity (cytotoxic activity) or antiproliferative action (cell proliferation inhibitory activity) against cells expressing a target antigen, or therapeutic methods that use the pharmaceutical compositions.BACKGROUND ART

[0003] To date, attempts have been made to develop a number of therapeutic vaccines directed to tumor cells. This is because it is considered that there are qualitative or quantitative differences between tumor cells and normal cells that may be recognized by the immune system of a living organism, and the immune system stimulated by active and specific sensitization by vaccines utilizing such differences (neoepitopes) can recognize and eliminate tumor cells.

[0004] To bring about such anti-tumor response, at least two conditions may have to be met. Firstly, the tumor cells must express an antigen that does not appear in normal cells, or express an antigen to such an extent that normal cells and tumor cells can be distinguished solely in a qualitative manner. Secondly, the immune system must be activated by vaccines or such in order to react with the antigen of interest. A major obstacle in tumor immunotherapy is considered to be that the immunogenicity of tumors is particularly weak in humans.

[0005] In recent years, tumor-related and tumor-specific antigens including such neoepitopes that may constitute targets to be attacked by the immune system have been discovered. Nonetheless, the immune system cannot eliminate tumors expressing such neoepitopes, and this may be due to insufficient immune response to these neoepitopes, rather than due to the absence of neoepitopes.

[0006] Two general strategies have been developed for the purpose of cell-based cancer immunotherapies. One of them is adoptive immunotherapy where tumor-reactive T lymphocytes expanded in vitro are reintroduced into a patient, and the other is active immunotherapy which uses tumor cells to induce systemic tumor response by triggering new or stronger immune response to a tumor antigen.

[0007] Tumor vaccines based on active immunotherapy have been prepared by various methods. To induce immune response to a tumor antigen, irradiated tumor cells mixed with an immune-stimulating adjuvant such as Bacillus Calmette Guerin (BCG) (Non-Patent Document 1), tumor cells genetically modified to produce, for example, cytokines (Non-Patent Document 2), and alienated autologous tumor cells (Non-Patent Document 3) have been prepared. However, the immunogenicity of the tumor cells is low, and this is considered to be due to the quantity of the tumor antigen, not the quality.

[0008] On the other hand, antibodies are known to induce humoral immune responses (production of antibodies against an antigen) and cellular immune responses (production of CD8-positive T cells against an antigen) to antigens by cross-presenting bound antigens to antigen-presenting cells, and it has been reported that administration of an antibody can induce acquired immunity to an antigen (Non-Patent Document 4). Recently, for the anti-tumor effect by an anti-HER2 antibody, it has been shown in an in vivo mouse model that acquired immunity to the HER2 antigen induced by administration of the antibody plays a more important role than the direct ADCC of the administered antibody (Non-Patent Document 5). In fact, in clinical use of Herceptin, which is an IgG1 subclass antibody drug against HER2, acquired immunity was induced by Herceptin administration, and humoral immune response to HER2 was observed (Non-Patent Document 6). Since patients in whom Herceptin administration was effective particularly showed an increased anti-HER2 antibody titer, induction of acquired immunity by Herceptin administration was considered to play an important role in the anti-tumor effect.

[0009] Antibodies are highly stable in blood and have few side effects, and are therefore drawing attention as pharmaceuticals (Non-Patent Documents 7 and 8). Many studies have been carried out so far on antibody-dependent cellular cytotoxicity (hereinafter denoted as ADCC) and complement-dependent cytotoxicity (hereinafter denoted as CDC), which are effector functions of IgG class antibodies. It has been reported that in the human IgG class, antibodies of the IgG1 subclass have the highest ADCC activity and CDC activity (Non-Patent Document 9). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG class antibodies, is also suggested to be one of the antibody effector functions (Non-Patent Documents 10 and 11). Since IgG1 subclass antibodies can exert these effector functions against tumors, IgG1 subclass antibodies are used for most antibody pharmaceuticals against cancer antigens.

[0010] In order for IgG antibodies to mediate ADCC and ADCP activities, the Fc region of the IgG antibodies must bind to antibody receptors (hereinafter denoted as FcγR) that are present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages. In humans, isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb have been reported as members of the FcγR protein family, and their respective allotypes have been reported as well (Non-Patent Document 12).

[0011] Enhancement of cytotoxic effector functions such as ADCC and ADCP has been drawing attention as a promising means for enhancing the antitumor effects of anticancer antibodies. Importance of FcγR-mediated effector functions aimed for antitumor effects of antibodies has been reported using mouse models (Non-Patent Documents 13 and 14). Furthermore, it was observed that clinical effects in humans correlated with the high-affinity polymorphic allotype (V158) and the low-affinity polymorphic allotype (F158) of FcγRIIIa (Non-Patent Document 15). These reports suggest that antibodies with an Fc region optimized for binding to a specific FcγR mediates stronger effector functions, and thereby exert more effective antitumor effects. The balance between the affinity of antibodies against the activating receptors including FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and the inhibitory receptors including FcγRIIb is an important factor in optimizing antibody effector functions. Enhancing the affinity to activating receptors may give antibodies a property to mediate stronger effector functions (Non-Patent Document 16), and therefore has been reported in various reports to date as an antibody engineering technique for improving or enhancing the antitumor activity of antibody pharmaceuticals against cancer antigens.

[0012] Regarding binding between the Fc region and FcγR, several amino acid residues in the antibody hinge region and the CH2 domain, and a sugar chain added to Asn at position 297 (EU numbering) bound to the CH2 domain have been shown as being important (Non-Patent Documents 9, 17, and 18). Focusing on this binding site, studies have so far been carried out on mutants of the Fc region having various FcγR binding properties, and Fc region mutants with higher affinity to activating FcγR have been obtained (Patent Documents 1 and 2). For example, Lazar et al. have succeeded in increasing the binding of human IgG1 to human FcγRIIIa (V158) by approximately 370 fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332 (EU numbering) of human IgG1 with Asn, Leu, and Glu, respectively (Non-Patent Document 19 and Patent Document 2). The ratio of binding to FcγRIIIa and FcγRIIb (A / I ratio) for this mutant was approximately 9-fold that of the wild type. Furthermore, Shinkawa et al. have succeeded in increasing the binding to FcγRIIIa up to approximately 100 fold by removing fucose from the sugar chain added to Asn at position 297 (EU numbering) (Non-Patent Document 20). These methods can greatly improve the ADCC activity of human IgG1 compared to that of naturally-occurring human IgG1.

[0013] While there are many reports, as described above, on methods for enhancing ADCC by antibody engineering, no reports have been made to date on antibody engineering techniques for enhancing or improving induction of acquired immunity by antibody administration. There is a report on methods for inducing acquired immunity against a cancer antigen, in which a cancer antigen against which acquired immunity is desired to be induced is fused with an antibody that binds to a high-mannose receptor or DEC-205 expressed on antigen presenting cells, thereby promoting incorporation and presentation of the cancer antigen by antigen presenting cells (Non-Patent Document 21). However, in these methods the target of antibody binding is not a cancer antigen as in the case of the above-mentioned anti-HER2 antibody. That is, since these methods induce acquired immunity against a cancer antigen fused to the antibody itself, the antibody itself cannot bind to the cancer antigen, and has the disadvantage of not being able to exhibit direct action on the cancer antigen. Furthermore, since this method induces acquired immunity not only against the cancer antigen fused to the antibody but also against the antibody itself used for targeting antigen-presenting cells, anti-drug antibodies will emerge and this leads to weakening of the effects. Therefore, this method may not be preferable for therapeutic purposes.

[0014] According to the above, while it is desirable to induce acquired immunity to a target antigen by administering an antigen-binding molecule having binding activity to the target antigen, there has been no reports on engineering techniques for improving or enhancing acquired immunity by such methods.PRIOR ART DOCUMENTSPatent Documents

[0015] [Patent Document 1] WO2000 / 042072

[0016] [Patent Document 2] WO2006 / 019447Non-Patent Documents

[0017] [Non-patent Document 1] Oettgen, H. F., and Old, L. J., The history of cancer immunotherapy, Biological Therapy of Cancer (1991) 87-119 DeVita et al. ed.

[0018] [Non-patent Document 2] Zatloukal K, Schmidt W, Cotten M, Wagner E, Stingl G, Birnstiel M L., Somatic gene therapy for cancer: the utility of transferrinfection in generating ‘tumor vaccines’, Gene (1993) 135, 199-207

[0019] [Non-patent Document 3] Bronte V, Tsung K, Rao J B, Chen P W, Wang M, Rosenberg S A, Restifo N P., IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases, J. Immunol. (1995) 154, 5282-5292

[0020] [Non-patent Document 4] Adams G P, Weiner L M., Monoclonal antibody therapy of cancer, Nat. Biotechnol. (2005) 23, 1147-1157

[0021] [Non-patent Document 5] Park S, Jiang Z, Mortenson E D, Deng L, Radkevich-Brown O, Yang X, Sattar H, Wang Y, Brown N K, Greene M, Liu Y, Tang J, Wang S, Fu Y X., The therapeutic effect of anti-HER2 / neu antibody depends on both innate and adaptive immunity, Cancer Cell (2010) 18, 160-170 (2010)

[0022] [Non-patent Document 6] Taylor C, Hershman D, Shah N, Suciu-Foca N, Petrylak D P, Taub R, Vandat L, Cheng B, Pegram M, Knutson K L, Clynes R. Augmented HER-2 specific immunity during treatment with trastuzumab and chemotherapy, Clin. Cancer Res, (2007) 13, 5133-43

[0023] [Non-patent Document 7] anice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic, Nat. Biotechnol. (2005) 23, 1073-1078

[0024] [Non-patent Document 8] avlou A K, Belsey M J., The therapeutic antibodies market to 2008, Eur. J. Pharm. Biopharm. (2005) 59(3), 389-396

[0025] [Non-patent Document 9] Clark, M., Antibody Engineering IgG Effector Mechanisms, Chemical Immunology (1997), 65, 88-110

[0026] [Non-patent Document 10] Horton H M, Bernett M J, Pong E, Peipp M, Karki S, Chu S Y, Richards J O, Vostiar I, Joyce P F, Repp R, Desjarlais J R, Zhukovsky E A., Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia, Cancer Res. (2008) 68, 8049-8057

[0027] [Non-patent Document 11] Zalevsky J, Leung I W, Karki S, Chu S Y, Zhukovsky E A, Desjarlais J R, Carmichael D F, Lawrence C E., The impact of Fc engineering on an anti-CD19 antibody: increased Fcγ receptor affinity enhances B-cell clearing in nonhuman primates, Blood (2009) 113, 3735-3743

[0028] [Non-patent Document 12] Jefferis R, Lund J., Interaction sites on human IgG-Fc for FcgammaR: current models, Immunol. Lett. (2002) 82, 57-65

[0029] [Non-patent Document 13] Clynes, R., Yoshizumi, T., Moroi, Y, Houghton, A. N., and Ravetch, J. V., Fc Receptors are required for passive and active immunity to melanoma, Proc. Natl. Acad. Sci. U.S.A (1998) 95, 652-656

[0030] [Non-patent Document 14] Clynes R A, Towers T L, Presta L C; Ravetch J V., Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets, Nat. Med. (2000) 6, 443-446

[0031] [Non-patent Document 15] Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H., Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene, Blood (2002) 99, 754-758

[0032] [Non-patent Document 16] Nimmerjahn F, Ravetch J V., Divergent immunoglobulin g subclass activity through selective Fc receptor binding, Science (2005), 310, 1510-1512

[0033] [Non-patent Document 17] Greenwood J, Clark M, Waldmann H., Structural motifs involved in human IgG antibody effector functions, Eur. J. Immunol. 23, 1098-1104 (1993)

[0034] [Non-patent Document 18] Morgan A, Jones N D, Nesbitt A M, Chaplin L, Bodmer M W, Emtage J S., The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding, Immunology (1995) 86, 319-324

[0035] [Non-patent Document 19] Lazar G A, Dang W, Karki S, Vafa O, Peng J S, Hyun L, Chan C, Chung H S, Eivazi A, Yoder S C, Vielmetter J, Carmichael D F, Hayes R J, Dahiyat B I., Engineered antibody Fc variants with enhanced effector function, Proc. Natl. Acad. Sci. U.S.A (2006) 103, 4005-4010

[0036] [Non-patent Document 20] Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K., The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity, J. Biol. Chem. (2003) 278, 3466-3473

[0037] [Non-patent Document 21] Tsuji T, Matsuzaki J, Kelly M P, Ramakrishna V, Vitale L, He L Z, Keler T, Odunsi K, Old L J, Ritter G, Gnjatic S., Antibody-Targeted NY-ESO-1 to Mannose Receptor or DEC-205 In Vitro Elicits Dual Human CD8+ and CD4+ T Cell Responses with Broad Antigen Specificity, J. Immunol. (2011) 186, 1218-1827SUMMARY OF THE INVENTIONProblems to be Solved by the Invention

[0038] The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide pharmaceutical compositions comprising as an active ingredient an antigen-binding molecule that induces an immune response in subjects affected with cancer or infected with foreign biological species when administered to these subjects, or therapeutic methods that use the pharmaceutical compositions. Another objective is to provide pharmaceutical compositions comprising as an active ingredient an antigen-binding molecule that induces the above-mentioned immune response and also has cytotoxicity (cytotoxic activity) or antiproliferative action (cell proliferation inhibitory activity) against cancer cells or infecting foreign biological species, or therapeutic methods that use the pharmaceutical compositions.Means for Solving the Problems

[0039] The present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose binding activity against an antigen changes depending on ion concentration conditions and containing an FcRn-binding domain having FcRn-binding activity under a neutral pH range, immune responses to the antigen are induced. Furthermore, the present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an FcRn-binding domain having FcRn-binding activity under a neutral pH range, immune responses to the antigen are induced, and the antigen-binding molecule can also have cytotoxicity or antiproliferative effect against cancer cells, foreign biological species, or the like that express the antigen to which the antigen-binding molecule binds. Based on these findings, the present inventors have elucidated that the antigen-binding molecules of the present invention are useful as pharmaceutical compositions for inducing an immune response in a subject infected with a foreign biological species or affected with cancer when administered to the subject. The present inventors have also elucidated that the antigen-binding molecules of the present invention are useful as pharmaceutical compositions that, when administered to a subject infected with a foreign biological species or affected with cancer, induce an immune response in the subject and also have cytotoxicity or antiproliferative effect against the cancer cells and foreign biological species. Methods for producing these pharmaceutical compositions have also been discovered.

[0040] More specifically, the present invention provides [1] to

[47] below:

[0041] [1] a pharmaceutical composition that induces an immune response to an antigen, which comprises as an active ingredient an antigen-binding molecule, wherein the antigen-binding molecule comprises an antigen-binding domain whose binding activity to the antigen changes depending on an ion concentration condition and comprises an FcRn-binding domain having binding activity to FcRn in a neutral pH range;

[0042] [2] the pharmaceutical composition of [1], wherein the ion concentration is a calcium ion concentration;

[0043] [3] the pharmaceutical composition of [2], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition;

[0044] [4] the pharmaceutical composition of [1], wherein the ion concentration condition is a pH condition;

[0045] [5] the pharmaceutical composition of [4], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity is higher in a neutral pH range than under an acidic pH range;

[0046] [6] the pharmaceutical composition of any one of [1] to [5], wherein the antigen-binding molecule has neutralizing activity against the antigen;

[0047] [7] the pharmaceutical composition of any one of [1] to [6], wherein the antigen-binding molecule has cytotoxic activity against a cell expressing the antigen;

[0048] [8] the pharmaceutical composition of any one of [1] to [7], wherein the FcRn-binding domain comprises an antibody Fc region;

[0049] [9] the pharmaceutical composition of [8], wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 257, 308, 428, and 434 according to EU numbering in the Fc region are different from amino acids at corresponding positions in a naturally-occurring Fc region;

[0050]

[10] the pharmaceutical composition of [8] or [9], wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:

[0051] Ala at amino acid position 257;

[0052] Pro at amino acid position 308;

[0053] Leu at amino acid position 428; and

[0054] Tyr at amino acid position 434,

[0055] according to EU numbering in the Fc region;

[0056]

[11] the pharmaceutical composition of any one of [8] to

[10] , wherein the Fcγ receptor-binding activity of the Fc region is higher than that of a naturally-occurring human IgG Fc region in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0057]

[12] the pharmaceutical composition of

[11] , wherein the Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);

[0058]

[13] the pharmaceutical composition of

[11] or

[12] , wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 according to EU numbering in the Fc region are different from amino acids at corresponding positions in a naturally-occurring Fc region;

[0059]

[14] the pharmaceutical composition of any one of

[11] to

[13] , wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:

[0060] either Lys or Tyr at amino acid position 221;

[0061] any one of Phe, Trp, Glu, and Tyr at amino acid position 222;

[0062] any one of Phe, Trp, Glu, and Lys at amino acid position 223;

[0063] any one of Phe, Trp, Glu, and Tyr at amino acid position 224;

[0064] any one of Glu, Lys, and Trp at amino acid position 225;

[0065] any one of Glu, Gly, Lys, and Tyr at amino acid position 227;

[0066] any one of Glu, Gly, Lys, and Tyr at amino acid position 228;

[0067] any one of Ala, Glu, Gly, and Tyr at amino acid position 230;

[0068] any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231;

[0069] any one of Glu, Gly, Lys, and Tyr at amino acid position 232;

[0070] any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 233;

[0071] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

[0072] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

[0073] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and

[0074] Tyr at amino acid position 236;

[0075] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

[0076] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 238;

[0077] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

[0078] any one of Ala, Ile, Met, and Thr at amino acid position 240;

[0079] any one of Asp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241;

[0080] any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position 243;

[0081] His at amino acid position 244;

[0082] Ala at amino acid position 245;

[0083] any one of Asp, Glu, His, and Tyr at amino acid position 246;

[0084] any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at amino acid position 247;

[0085] any one of Glu, His, Gln, and Tyr at amino acid position 249;

[0086] either Glu or Gln at amino acid position 250;

[0087] Phe at amino acid position 251;

[0088] any one of Phe, Met, and Tyr at amino acid position 254;

[0089] any one of Glu, Leu, and Tyr at amino acid position 255;

[0090] any one of Ala, Met, and Pro at amino acid position 256;

[0091] any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258;

[0092] any one of Asp, Glu, His, and Tyr at amino acid position 260;

[0093] any one of Ala, Glu, Phe, Ile, and Thr at amino acid position 262;

[0094] any one of Ala, Ile, Met, and Thr at amino acid position 263;

[0095] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 264;

[0096] any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr at amino acid position 265;

[0097] any one of Ala, Ile, Met, and Thr at amino acid position 266;

[0098] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

[0099] any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

[0100] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 269;

[0101] any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 270;

[0102] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;

[0103] any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 272;

[0104] either Phe or Ile at amino acid position 273;

[0105] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

[0106] either Leu or Trp at amino acid position 275;

[0107] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 276;

[0108] any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

[0109] Ala at amino acid position 279;

[0110] any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acid position 280;

[0111] any one of Asp, Lys, Pro, and Tyr at amino acid position 281;

[0112] any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282;

[0113] any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at amino acid position 283;

[0114] any one of Asp, Glu, Leu, Asn, Thr, and Tyr at amino acid position 284;

[0115] any one of Asp, Glu, Lys, Gln, Trp, and Tyr at amino acid position 285;

[0116] any one of Glu, Gly, Pro, and Tyr at amino acid position 286;

[0117] any one of Asn, Asp, Glu, and Tyr at amino acid position 288;

[0118] any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at amino acid position 290;

[0119] any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at amino acid position 291;

[0120] any one of Ala, Asp, Glu, Pro, Thr, and Tyr at amino acid position 292;

[0121] any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 293;

[0122] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 294;

[0123] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

[0124] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

[0125] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

[0126] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 298;

[0127] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at amino acid position 299;

[0128] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300;

[0129] any one of Asp, Glu, His, and Tyr at amino acid position 301;

[0130] Ile at amino acid position 302;

[0131] any one of Asp, Gly, and Tyr at amino acid position 303;

[0132] any one of Asp, His, Leu, Asn, and Thr at amino acid position 304;

[0133] any one of Glu, Ile, Thr, and Tyr at amino acid position 305;

[0134] any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position 311;

[0135] Phe at amino acid position 313;

[0136] Leu at amino acid position 315;

[0137] either Glu or Gln at amino acid position 317;

[0138] any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acid position 318;

[0139] any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 320;

[0140] any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 322;

[0141] Ile at amino acid position 323;

[0142] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position 324;

[0143] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

[0144] any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at amino acid position 326;

[0145] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position 327;

[0146] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

[0147] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

[0148] any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

[0149] any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 331;

[0150] any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

[0151] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

[0152] any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position 334;

[0153] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at amino acid position 335;

[0154] any one of Glu, Lys, and Tyr at amino acid position 336;

[0155] any one of Glu, His, and Asn at amino acid position 337;

[0156] any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at amino acid position 339;

[0157] either Ala or Val at amino acid position 376;

[0158] either Gly or Lys at amino acid position 377;

[0159] Asp at amino acid position 378;

[0160] Asn at amino acid position 379;

[0161] any one of Ala, Asn, and Ser at amino acid position 380;

[0162] either Ala or Ile at amino acid position 382;

[0163] Glu at amino acid position 385;

[0164] Thr at amino acid position 392;

[0165] Leu at amino acid position 396;

[0166] Lys at amino acid position 421;

[0167] Asn at amino acid position 427;

[0168] either Phe or Leu at amino acid position 428;

[0169] Met at amino acid position 429;

[0170] Trp at amino acid position 434;

[0171] Ile at amino acid position 436; and

[0172] any one of Gly, His, Ile, Leu, and Tyr at amino acid position 440;

[0173] according to EU numbering in the Fc region;

[0174]

[15] the pharmaceutical composition of any one of

[11] to

[14] , wherein the naturally-occurring Fc region is an Fc region of any one of human IgG1, human IgG2, human IgG3, and human IgG4 in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0175]

[16] the pharmaceutical composition of any one of

[11] to

[15] , wherein the Fc region is modified so that the percentage of the Fc region to which a fucose-deficient sugar chain is attached, or bisecting N-acetylglucosamine is added, at position 297 according to EU numbering in the Fc region, will become higher.

[0176]

[17] a method for inducing an immune response in a living organism, which comprises the step of administering the antigen-binding molecule of any one of [1] to

[16] to the living organism;

[0177]

[18] a method for producing an antigen-binding molecule that induces an immune response, which comprises imparting FcRn-binding activity in a neutral pH range to an FcRn-binding domain that is contained in an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on an ion concentration condition;

[0178]

[19] the method of

[18] , wherein the ion concentration is a calcium ion concentration;

[0179]

[20] the method of

[19] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition;

[0180]

[21] the method of

[18] , wherein the ion concentration condition is a pH condition;

[0181]

[22] the method of

[21] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity is higher in a neutral pH range than in an acidic pH range;

[0182]

[23] the method of any one of

[18] to

[22] , wherein the antigen-binding molecule has neutralizing activity against the antigen;

[0183]

[24] the method of any one of

[18] to

[23] , wherein the antigen-binding molecule has cytotoxic activity against a cell expressing the antigen;

[0184]

[25] the method of any one of

[18] to

[24] , wherein the FcRn-binding domain comprises an antibody Fc region;

[0185]

[26] the method of

[25] , comprising the step of substituting at least one or more amino acids selected from the group consisting of amino acids at positions 239, 252, 257, 286, 307, 308, 428, and 434 according to EU numbering in the Fc region;

[0186]

[27] the method of

[25] or

[26] , comprising the step of performing at least one or more amino acid substitutions selected from the group consisting of:

[0187] amino acid substitution with Ala at position 257;

[0188] amino acid substitution with Pro at position 308;

[0189] amino acid substitution with Leu at position 428; and

[0190] amino acid substitution with Tyr at position 434,

[0191] according to EU numbering in the Fc region;

[0192]

[28] the method of any one of

[25] to

[27] , comprising the step of enhancing the Fcγ receptor-binding activity of the Fc region as compared to that of a naturally-occurring human IgG Fc region in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0193]

[29] the method of

[28] , wherein the Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);

[0194]

[30] the method of

[28] or

[29] , comprising the step of substituting at least one or more amino acids selected from the group consisting of amino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 according to EU numbering in the Fc region;

[0195]

[31] the method of any one of

[28] to

[30] , comprising the step of performing at least one or more amino acid substitutions selected from the group consisting of:

[0196] amino acid substitution with either Lys or Tyr at position 221;

[0197] amino acid substitution with any one of Phe, Trp, Glu, and Tyr at position 222;

[0198] amino acid substitution with any one of Phe, Trp, Glu, and Lys at position 223;

[0199] amino acid substitution with any one of Phe, Trp, Glu, and Tyr at position 224;

[0200] amino acid substitution with any one of Glu, Lys, and Trp at position 225;

[0201] amino acid substitution with any one of Glu, Gly, Lys, and Tyr at position 227;

[0202] amino acid substitution with any one of Glu, Gly, Lys, and Tyr at position 228;

[0203] amino acid substitution with any one of Ala, Glu, Gly, and Tyr at position 230;

[0204] amino acid substitution with any one of Glu, Gly, Lys, Pro, and Tyr at position 231;

[0205] amino acid substitution with any one of Glu, Gly, Lys, and Tyr at position 232;

[0206] amino acid substitution with any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,

[0207] Arg, Ser, Thr, Val, Trp, and Tyr at position 233;

[0208] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 234;

[0209] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 235;

[0210] amino acid substitution with any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 236;

[0211] amino acid substitution with any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 237;

[0212] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 238;

[0213] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 239;

[0214] amino acid substitution with any one of Ala, Ile, Met, and Thr at position 240;

[0215] amino acid substitution with any one of Asp, Glu, Leu, Arg, Trp, and Tyr at position 241;

[0216] amino acid substitution with any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at position 243;

[0217] amino acid substitution with His at position 244;

[0218] amino acid substitution with Ala at position 245;

[0219] amino acid substitution with any one of Asp, Glu, His, and Tyr at position 246;

[0220] amino acid substitution with any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at position 247;

[0221] amino acid substitution with any one of Glu, His, Gln, and Tyr at position 249;

[0222] amino acid substitution with either Glu or Gln at position 250;

[0223] amino acid substitution with Phe at position 251;

[0224] amino acid substitution with any one of Phe, Met, and Tyr at position 254;

[0225] amino acid substitution with any one of Glu, Leu, and Tyr at position 255;

[0226] amino acid substitution with any one of Ala, Met, and Pro at position 256;

[0227] amino acid substitution with any one of Asp, Glu, His, Ser, and Tyr at position 258;

[0228] amino acid substitution with any one of Asp, Glu, His, and Tyr at position 260;

[0229] amino acid substitution with any one of Ala, Glu, Phe, Ile, and Thr at position 262;

[0230] amino acid substitution with any one of Ala, Ile, Met, and Thr at position 263;

[0231] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 264;

[0232] amino acid substitution with any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr at position 265;

[0233] amino acid substitution with any one of Ala, Ile, Met, and Thr at position 266;

[0234] amino acid substitution with any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 267;

[0235] amino acid substitution with any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at position 268;

[0236] amino acid substitution with any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,

[0237] Val, Trp, and Tyr at position 269;

[0238] amino acid substitution with any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 270;

[0239] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 271;

[0240] amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr,

[0241] Val, Trp, and Tyr at position 272;

[0242] amino acid substitution with either Phe or Ile at position 273;

[0243] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 274;

[0244] amino acid substitution with either Leu or Trp at position 275;

[0245] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 276;

[0246] amino acid substitution with any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 278;

[0247] amino acid substitution with Ala at position 279;

[0248] amino acid substitution with any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at position 280;

[0249] amino acid substitution with any one of Asp, Lys, Pro, and Tyr at position 281;

[0250] amino acid substitution with any one of Glu, Gly, Lys, Pro, and Tyr at position 282;

[0251] amino acid substitution with any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at position 283;

[0252] amino acid substitution with at position 284 is any one of Asp, Glu, Leu, Asn, Thr, and Tyr;

[0253] amino acid substitution with any one of Asp, Glu, Lys, Gln, Trp, and Tyr at position 285;

[0254] amino acid substitution with any one of Glu, Gly, Pro, and Tyr at position 286;

[0255] amino acid substitution with any one of Asn, Asp, Glu, and Tyr at position 288;

[0256] amino acid substitution with any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at position 290;

[0257] amino acid substitution with any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at position 291;

[0258] amino acid substitution with any one of Ala, Asp, Glu, Pro, Thr, and Tyr at position 292;

[0259] amino acid substitution with any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,

[0260] Trp, and Tyr at position 293;

[0261] amino acid substitution with any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 294;

[0262] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 295;

[0263] amino acid substitution with any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at position 296;

[0264] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 297;

[0265] amino acid substitution with any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at position 298;

[0266] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at position 299;

[0267] amino acid substitution with any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 300;

[0268] amino acid substitution with any one of Asp, Glu, His, and Tyr at position 301;

[0269] amino acid substitution with Ile at position 302;

[0270] amino acid substitution with any one of Asp, Gly, and Tyr at position 303;

[0271] amino acid substitution with at position 304 is any one of Asp, His, Leu, Asn, and Thr;

[0272] amino acid substitution with any one of Glu, Ile, Thr, and Tyr at position 305;

[0273] amino acid substitution with any one of Ala, Asp, Asn, Thr, Val, and Tyr at position 311;

[0274] amino acid substitution with Phe at position 313;

[0275] amino acid substitution with Leu at position 315;

[0276] amino acid substitution with either Glu or Gln at position 317;

[0277] amino acid substitution with any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at position 318;

[0278] amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at position 320;

[0279] amino acid substitution with any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at position 322;

[0280] amino acid substitution with Ile at position 323;

[0281] amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at position 324;

[0282] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 325;

[0283] amino acid substitution with any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr,

[0284] Val, Trp, and Tyr at position 326;

[0285] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at position 327;

[0286] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 328;

[0287] amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 329;

[0288] amino acid substitution with any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 330;

[0289] amino acid substitution with any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at position 331;

[0290] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 332;

[0291] amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at position 333;

[0292] amino acid substitution with any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at position 334;

[0293] amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at position 335;

[0294] amino acid substitution with any one of Glu, Lys, and Tyr at position 336;

[0295] amino acid substitution with any one of Glu, His, and Asn at position 337;

[0296] amino acid substitution with any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at position 339;

[0297] amino acid substitution with either Ala or Val at position 376;

[0298] amino acid substitution with either Gly or Lys at position 377;

[0299] amino acid substitution with Asp at position 378;

[0300] amino acid substitution with Asn at position 379;

[0301] amino acid substitution with any one of Ala, Asn, and Ser at position 380;

[0302] amino acid substitution with either Ala or Ile at position 382;

[0303] amino acid substitution with Glu at position 385;

[0304] amino acid substitution with Thr at position 392;

[0305] amino acid substitution with Leu at position 396;

[0306] amino acid substitution with Lys at position 421;

[0307] amino acid substitution with Asn at position 427;

[0308] amino acid substitution with either Phe or Leu at position 428;

[0309] amino acid substitution with Met at position 429;

[0310] amino acid substitution with Trp at position 434;

[0311] amino acid substitution with Ile at position 436; and

[0312] amino acid substitution with any one of Gly, His, Ile, Leu, and Tyr at position 440,

[0313] according to EU numbering in the Fc region;

[0314]

[32] the method of any one of

[28] to

[31] , wherein the naturally-occurring Fc region is an Fc region of any one of human IgG1, human IgG2, human IgG3, and human IgG4 in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0315]

[33] the method of any one of

[28] to

[32] , comprising the step of modifying the Fc region so that the percentage of the Fc region to which a fucose-deficient sugar chain is attached, or bisecting N-acetylglucosamine is added, at position 297 according to EU numbering in the Fc region, will become higher;

[0316]

[34] a method for producing a pharmaceutical composition which induces an immune response, which comprises the steps of:

[0317] (a) determining the antigen-binding activity of an antigen-binding domain under a high calcium ion concentration condition;

[0318] (b) determining the antigen-binding activity of the antigen-binding domain under a low calcium ion concentration condition;

[0319] (c) selecting the antigen-binding domain whose antigen-binding activity determined in (a) is higher than that determined in (b);

[0320] (d) linking a polynucleotide encoding the antigen-binding domain selected in (c) to a polynucleotide encoding an FcRn-binding domain having FcRn-binding activity in a neutral pH range;

[0321] (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and

[0322] (f) collecting an antigen-binding molecule from culture fluid of the cell cultured in (e);

[0323]

[35] a method for producing a pharmaceutical composition which induces an immune response, which comprises the steps of:

[0324] (a) determining the antigen-binding activity of an antibody under a high calcium ion concentration condition;

[0325] (b) determining the antigen-binding activity of the antibody under a low calcium ion concentration condition;

[0326] (c) selecting the antibody whose antigen-binding activity determined in (a) is higher than that determined in (b);

[0327] (d) linking a polynucleotide encoding the antigen-binding domain of the antibody selected in (c) to a polynucleotide encoding an FcRn-binding domain having FcRn-binding activity in a neutral pH range;

[0328] (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and

[0329] (f) collecting an antigen-binding molecule from culture fluid of the cell cultured in (e);

[0330]

[36] a method for producing an antigen-binding molecule, which comprises the steps of:

[0331] (a) determining the antigen-binding activity of an antigen-binding domain in a neutral pH range;

[0332] (b) determining the antigen-binding activity of the antigen-binding domain in an acidic pH range;

[0333] (c) selecting the antigen-binding domain whose antigen-binding activity determined in (a) is higher than that determined in (b);

[0334] (d) linking a polynucleotide encoding the antigen-binding domain selected in (c) to a polynucleotide encoding an FcRn-binding domain having FcRn-binding activity in a neutral pH range;

[0335] (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and

[0336] (f) collecting an antigen-binding molecule from culture fluid of the cell cultured in (e);

[0337]

[37] a method for producing an antigen-binding molecule, which comprises the steps of:

[0338] (a) determining the antigen-binding activity of an antibody in a neutral pH range;

[0339] (b) determining the antigen-binding activity of the antibody in an acidic pH range;

[0340] (c) selecting the antibody whose antigen-binding activity determined in (a) is higher than that determined in (b);

[0341] (d) linking a polynucleotide encoding the antigen-binding domain of the antibody selected in (c) to a polynucleotide encoding an FcRn-binding domain having FcRn-binding activity in a neutral pH range;

[0342] (e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked has been introduced; and

[0343] (f) collecting an antigen-binding molecule from culture fluid of the cell cultured in (e);

[0344]

[38] the method of any one of

[34] to

[37] , wherein the antigen-binding molecule has neutralizing activity against the antigen;

[0345]

[39] the method of any one of

[34] to

[38] , wherein the antigen-binding molecule has cytotoxic activity against a cell expressing the antigen;

[0346]

[40] the method of any one of

[34] to

[39] , wherein the FcRn-binding domain comprises an antibody Fc region;

[0347]

[41] the method of

[40] , wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of amino acids at positions 257, 308, 428, and 434 according to EU numbering in the Fc region are different from amino acids at corresponding positions in a naturally-occurring Fc region;

[0348]

[42] the method of

[40] or

[41] , wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:

[0349] Ala at amino acid position 257;

[0350] Pro at amino acid position 308;

[0351] Leu at amino acid position 428; and

[0352] Tyr at amino acid position 434,

[0353] according to EU numbering in the Fc region;

[0354]

[43] the method of any one of

[40] to

[42] , wherein the Fcγ receptor-binding activity of the Fc region is higher than that of a naturally-occurring human IgG Fc region in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0355]

[44] the method of

[43] , wherein the Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);

[0356]

[45] the method of

[43] or

[44] , wherein the Fc region comprises at least one or more amino acids selected from the group consisting of:

[0357] either Lys or Tyr at amino acid position 221;

[0358] any one of Phe, Trp, Glu, and Tyr at amino acid position 222;

[0359] any one of Phe, Trp, Glu, and Lys at amino acid position 223;

[0360] any one of Phe, Trp, Glu, and Tyr at amino acid position 224;

[0361] any one of Glu, Lys, and Trp at amino acid position 225;

[0362] any one of Glu, Gly, Lys, and Tyr at amino acid position 227;

[0363] any one of Glu, Gly, Lys, and Tyr at amino acid position 228;

[0364] any one of Ala, Glu, Gly, and Tyr at amino acid position 230;

[0365] any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231;

[0366] any one of Glu, Gly, Lys, and Tyr at amino acid position 232;

[0367] any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 233;

[0368] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;

[0369] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

[0370] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;

[0371] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;

[0372] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 238;

[0373] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;

[0374] any one of Ala, Ile, Met, and Thr at amino acid position 240;

[0375] any one of Asp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241;

[0376] any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position 243;

[0377] His at amino acid position 244;

[0378] Ala at amino acid position 245;

[0379] any one of Asp, Glu, His, and Tyr at amino acid position 246;

[0380] any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at amino acid position 247;

[0381] any one of Glu, His, Gln, and Tyr at amino acid position 249;

[0382] either Glu or Gln at amino acid position 250;

[0383] Phe at amino acid position 251;

[0384] any one of Phe, Met, and Tyr at amino acid position 254;

[0385] any one of Glu, Leu, and Tyr at amino acid position 255;

[0386] any one of Ala, Met, and Pro at amino acid position 256;

[0387] any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258;

[0388] any one of Asp, Glu, His, and Tyr at amino acid position 260;

[0389] any one of Ala, Glu, Phe, Ile, and Thr at amino acid position 262;

[0390] any one of Ala, Ile, Met, and Thr at amino acid position 263;

[0391] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 264;

[0392] any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr at amino acid position 265;

[0393] any one of Ala, Ile, Met, and Thr at amino acid position 266;

[0394] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267;

[0395] any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268;

[0396] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 269;

[0397] any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 270;

[0398] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;

[0399] any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 272;

[0400] either Phe or Ile at amino acid position 273;

[0401] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 274;

[0402] either Leu or Trp at amino acid position 275;

[0403] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 276;

[0404] any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 278;

[0405] Ala at amino acid position 279;

[0406] any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acid position 280;

[0407] any one of Asp, Lys, Pro, and Tyr at amino acid position 281;

[0408] any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282;

[0409] any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at amino acid position 283;

[0410] any one of Asp, Glu, Leu, Asn, Thr, and Tyr at amino acid position 284;

[0411] any one of Asp, Glu, Lys, Gln, Trp, and Tyr at amino acid position 285;

[0412] any one of Glu, Gly, Pro, and Tyr at amino acid position 286;

[0413] any one of Asn, Asp, Glu, and Tyr at amino acid position 288;

[0414] any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at amino acid position 290;

[0415] any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at amino acid position 291;

[0416] any one of Ala, Asp, Glu, Pro, Thr, and Tyr at amino acid position 292;

[0417] any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 293;

[0418] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 294;

[0419] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 295;

[0420] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296;

[0421] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 297;

[0422] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 298;

[0423] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at amino acid position 299;

[0424] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300;

[0425] any one of Asp, Glu, His, and Tyr at amino acid position 301;

[0426] Ile at amino acid position 302;

[0427] any one of Asp, Gly, and Tyr at amino acid position 303;

[0428] any one of Asp, His, Leu, Asn, and Thr at amino acid position 304;

[0429] any one of Glu, Ile, Thr, and Tyr at amino acid position 305;

[0430] any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position 311;

[0431] Phe at amino acid position 313;

[0432] Leu at amino acid position 315;

[0433] either Glu or Gln at amino acid position 317;

[0434] any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acid position 318;

[0435] any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 320;

[0436] any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 322;

[0437] Ile at amino acid position 323;

[0438] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position 324;

[0439] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;

[0440] any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at amino acid position 326;

[0441] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position 327;

[0442] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;

[0443] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 329;

[0444] any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;

[0445] any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 331;

[0446] any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;

[0447] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333;

[0448] any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position 334;

[0449] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at amino acid position 335;

[0450] any one of Glu, Lys, and Tyr at amino acid position 336;

[0451] any one of Glu, His, and Asn at amino acid position 337;

[0452] any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at amino acid position 339;

[0453] either Ala or Val at amino acid position 376;

[0454] either Gly or Lys at amino acid position 377;

[0455] Asp at amino acid position 378;

[0456] Asn at amino acid position 379;

[0457] any one of Ala, Asn, and Ser at amino acid position 380;

[0458] either Ala or Ile at amino acid position 382;

[0459] Glu at amino acid position 385;

[0460] Thr at amino acid position 392;

[0461] Leu at amino acid position 396;

[0462] Lys at amino acid position 421;

[0463] Asn at amino acid position 427;

[0464] either Phe or Leu at amino acid position 428;

[0465] Met at amino acid position 429;

[0466] Trp at amino acid position 434;

[0467] Ile at amino acid position 436; and

[0468] any one of Gly, His, Ile, Leu, and Tyr at amino acid position 440;

[0469] according to EU numbering in the Fc region;

[0470]

[46] the method of any one of

[43] to

[45] , wherein the naturally-occurring Fc region is an Fc region of any one of human IgG1, human IgG2, human IgG3, and human IgG4 in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain;

[0471]

[47] the method of any one of

[43] to

[46] , wherein the Fc region is modified so that the percentage of the Fc region to which a fucose-deficient sugar chain is attached, or bisecting N-acetylglucosamine is added, at position 297 according to EU numbering in the Fc region, will become higher.Effects of the Invention

[0472] The present invention provides pharmaceutical compositions comprising an antigen-binding molecule that, when administered to a living organism, can not only exhibit pharmacological actions on a target antigen but also induce an immune response to the target antigen, which was not possible with conventional antibodies, and provides methods for manufacturing them. This enables effective treatment of cancer and infectious diseases by induction of immune response to a target antigen while having binding activity to the target antigen and having cytotoxicity and antiproliferative activity against target cells, which was not possible with conventional vaccines.BRIEF DESCRIPTION OF THE DRAWINGS

[0473] FIG. 1 shows changes in the concentration of soluble human IL-6 receptor in mice plasma for the anti-mouse CD4 antibody administration group and the non-administration group in a soluble human IL-6 receptor steady-state model. The horizontal axis shows the number of days from anti-mouse CD4 antibody administration, and the vertical axis shows the plasma concentration of soluble human IL-6 receptor.

[0474] FIG. 2 shows changes in the concentration of soluble human IL-6 receptor in mice plasma for the normal anti-IL-6 receptor antibody and pH-dependent IL-6 receptor antibody administration groups in a human IL-6 receptor immunotolerance normal mouse model. The horizontal axis shows the number of days from anti-IL-6 receptor antibody administration, and the vertical axis shows the plasma concentration of soluble human IL-6 receptor. The filled circles indicate the plasma concentration of soluble human IL-6 receptor in control mice. The open circles indicate the plasma concentration of soluble human IL-6 receptor in H54 / L28-IgG1-administered mice, and the diamonds indicate the plasma concentration of soluble human IL-6 receptor in Fv4-IgG1-administered mice.

[0475] FIG. 3 shows changes in the concentration of soluble human IL-6 receptor in mice for the group to which normal anti-IL-6 receptor antibody with enhanced FcRn binding at pH7.4 was administered and the group to which pH-dependent IL-6 receptor antibody with enhanced FcRn binding at pH7.4 was administered, in a human IL-6 receptor immunotolerance normal mouse model. The horizontal axis shows the number of days from anti-IL-6 receptor antibody administration, and the vertical axis shows the plasma concentration of soluble human IL-6 receptor. The filled circles indicate the plasma concentration of soluble human IL-6 receptor in control mice. The plasma concentration of soluble human IL-6 receptor in mice to which H54 / L28-IgG1, Fv4-IgG1, H54 / L28-F157, or Fv4-F157 was administered is shown by the open circles, diamonds, open triangles, or X and the filled squares, respectively.

[0476] FIG. 4 shows a non-limiting action model of an ion-concentration-dependent antigen-binding molecule with regard to the lysosome transport of a soluble antigen. Under the plasma ion concentration conditions (in the neutral pH range or under high calcium ion concentration), the antigen-binding molecule that has bound to a soluble antigen in plasma (A) is taken up into a cell by non-specific endocytosis and such (B), and then is transferred to an acidic endosome where it binds to FcRn expressed in the endosome via the FcRn binding domain under the acidic pH condition, and releases the antigen under endosomal ion concentration conditions (in the acidic pH range or under low calcium ion concentration) (C). The released antigen is transferred to the lysosome and then degraded (D). On the other hand, the antigen-binding molecule that has released the antigen moves to the cell surface while being bound to FcRn, dissociates from FcRn under the neutral pH condition in the plasma, and then returns to the plasma (E).

[0477] FIG. 5 shows a non-limiting action model of an antigen-binding molecule having FcRn-binding activity at pH7.4 with regard to the lysosome transport of a soluble antigen. Under the plasma ion concentration conditions (in the neutral pH range or under high calcium ion concentration), the antigen-binding molecule which has bound to a soluble antigen in plasma binds to FcRn under the neutral pH condition via the FcRn-binding domain (A), and this is then taken up into the cell by endocytosis (B). The antigen-binding molecule that has transferred to an acidic endosome does not release the antigen under endosomal ion concentration conditions (in the acidic pH range or under low calcium ion concentration) (C), and the antigen-bound antigen-binding molecule is recycled onto the cell surface while being bound to FcRn (D).

[0478] FIG. 6 shows a non-limiting action model of an ion-concentration-dependent antigen-binding molecule with enhanced FcRn binding at pH7.4 with respect to lysosome transport of a soluble antigen. Under the plasma ion concentration conditions (in the neutral pH range or under high calcium ion concentration), the antigen-binding molecule which has bound to a soluble antigen in plasma binds to FcRn under neutral pH conditions via the FcRn-binding domain (A), and this is then taken up into the cell by endocytosis (B). The antigen-binding molecule that has transferred to an acidic endosome releases the antigen under endosomal ion concentration conditions (in the acidic pH range or under low calcium ion concentration) (C). The dissociated antigen is transferred to the lysosome and then degraded (D). On the other hand, the antigen-binding molecule that has released the antigen is recycled onto the cell surface while being bound to FcRn (E).

[0479] FIG. 7 shows changes in the plasma concentration of soluble human IL-6 receptor and changes in the titer of mouse anti-human IL-6 receptor antibody in each of the three Fv4-F157-administered mice (#7, 8, and 9) in Test 1. The horizontal axis shows the number of days from anti-IL-6 receptor antibody administration, the left vertical axis shows the concentration of soluble human IL-6 receptor in plasma, and the right vertical axis shows the ECL value, which serves as the indicator for the mouse anti-hsIL-6R antibody titer. The solid lines show the soluble human IL-6 receptor concentration in plasma and the dashed lines show the ECL values. The diamonds, open squares, and triangles show changes in the plasma concentration of soluble human IL-6 receptor of individuals #7, 8, and 9, respectively; and X, filled squares, and filled circles show changes in the ECL value in individuals #7, 8, and 9, respectively.

[0480] FIG. 8 shows changes in the plasma concentration of soluble human IL-6 receptor and changes in the titer of mouse anti-human IL-6 receptor antibody in each of the three Fv4-F157-administered mice (#10, 11, and 12) of Test 2. The horizontal axis shows the number of days from anti-IL-6 receptor antibody administration, the left vertical axis shows the concentration of soluble human IL-6 receptor in plasma, and the right vertical axis shows the ECL value, which serves as the indicator for the mouse anti-human IL-6 receptor antibody titer. The solid lines show the soluble human IL-6 receptor concentration in plasma and the dashed lines show the ECL values. The diamonds, open squares, and triangles show the concentration of soluble human IL-6 receptor in the plasma of individuals #10, 11, and 12, respectively; and X, filled squares, and filled circles show changes in the ECL value in individuals #10, 11, and 12, respectively.

[0481] FIG. 9 shows changes in the titer of mouse anti-hsIL-6R antibody and mouse anti-Fv4-F157 antibody in each of the three Fv4-F157-administered mice (#7, 8, and 9) of Test 1. The horizontal axis shows the number of days from the administration of anti-IL-6 receptor antibody, and the vertical axis shows the ECL values, which serve as the indicators for the mouse anti-human IL-6 receptor antibody titer and mouse anti-Fv4-F157 antibody titer. The solid lines show changes in the ECL value serving as the indicator for mouse anti-Fv4-F157 antibody titer, and the dashed lines show changes in the ECL value serving as the indicator for mouse anti-human IL-6 receptor antibody titer. The diamonds, open squares, and filled triangles show changes in the ECL value serving as the indicator for mouse anti-Fv4-F157 antibody titer in individuals #7, 8, and 9, respectively; and the open squares, filled squares, and open triangles show changes in the ECL value serving as the indicator for the mouse anti-human IL-6 receptor antibody titer in individuals #7, 8, and 9, respectively.

[0482] FIG. 10 shows changes in the titer of mouse anti-human IL-6 receptor antibody and changes in the titer of mouse anti-Fv4-F157 antibody in each of the three Fv4-F157-administered mice (#10, 11, and 12) of Test 2. The horizontal axis shows the number of days from the administration of anti-IL-6 receptor antibody, and the vertical axis shows the ECL values serving as the indicator for the mouse anti-human IL-6 receptor antibody titer and the indicator for the mouse anti-Fv4-F157 antibody titer. The solid lines show changes in the ECL value serving as the indicator for mouse anti-Fv4-F157 antibody titer, and the dashed lines show changes in the ECL value serving as the indicator for mouse anti-hsIL-6R antibody titer. The diamonds, open squares, and filled triangles show changes in the ECL value serving as the indicator for mouse anti-Fv4-F157 antibody titer in individuals #10, 11, and 12, respectively; and the open squares, filled squares, and open triangles show changes in the ECL value serving as the indicator for the mouse anti-human IL-6 receptor antibody titer in individuals #10, 11, and 12, respectively.

[0483] FIG. 11 depicts a non-limiting model of the mechanism of action of an antibody fused with a target antigen on cancer cells and antigen-presenting cells.

[0484] FIG. 12 depicts a non-limiting model of the mechanism of action of an antigen-binding molecule on cancer cells and antigen-presenting cells, where the antigen-binding molecule has FcRn-binding activity in the neutral pH range and has ion concentration-dependent binding activity to a target antigen.

[0485] FIG. 13 depicts the manner of interaction between an antigen and a calcium-dependent binding antibody in plasma (2 mM CO and in endosome (3 μM Ca′) (i), and the manner of interaction between an antigen and a pH- and calcium-dependent binding antibody in plasma (pH7.4, 2 mM CO and in endosome (pH6.0, 3 μM (ii).

[0486] FIG. 14 shows an ion-exchange chromatogram for an antibody comprising a human Vk5-2 sequence and an antibody comprising an hVk5-2_L65 sequence which has a modified glycosylation sequence of the human Vk5-2 sequence. The solid line represents a chromatogram for the antibody comprising the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 45; and light chain: hVk5-2, SEQ ID NO: 50). The broken line represents a chromatogram for the antibody comprising the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 45); and light chain: hVk5-2_L65 (SEQ ID NO: 53)).

[0487] FIG. 15 shows the relationship of a designed amino acid distribution (indicated as Design) to the amino acid distribution (indicated as Library) for the sequence information on 290 clones isolated from E. coli introduced with a gene library of antibodies that bind to antigens in a Ca-dependent manner. The horizontal axis indicates amino acid positions in the Kabat numbering system. The vertical axis indicates % amino acid distribution.

[0488] FIG. 16 shows sensorgrams for anti-IL-6R antibody (tocilizumab), antibody 6RC1IgG_010, antibody 6RC1IgG_012, and antibody 6RC1IgG_019 under a high calcium ion concentration (1.2 mM) condition.

[0489] FIG. 17 shows sensorgrams for anti-IL-6R antibody (tocilizumab), antibody 6RC1IgG_010, antibody 6RC1IgG_012, and antibody 6RC1IgG_019 under a low calcium ion concentration (3 μM) condition.

[0490] FIG. 18 depicts the structure of heavy-chain CDR3 of an Fab fragment of antibody 6RL #9 determined by X-ray crystallography. The heavy-chain CDR3 portion in the crystal structure obtained by crystallization in the presence of calcium ions is shown in (i), and the heavy-chain CDR3 portion in the crystal structure obtained by crystallization in the absence of calcium ions is shown in (ii).

[0491] FIG. 19 shows changes in plasma antibody concentrations in normal mice for the H54 / L28-IgG1 antibody, the FH4-IgG1 antibody, and the 6RL #9-IgG1 antibody.

[0492] FIG. 20 shows changes in the plasma concentration of soluble human IL-6 receptor (hsIL-6R) in normal mice for the H54 / L28-IgG1 antibody, the FH4-IgG1 antibody, and the 6RL #9-IgG1 antibody.

[0493] FIG. 21 shows changes in the plasma antibody concentrations in normal mice for the H54 / L28-N434W antibody, the FH4-N434W antibody, and the 6RL #9-N434W antibody.

[0494] FIG. 22 shows changes in the plasma concentration of soluble human IL-6 receptor (hsIL-6R) in normal mice for the H54 / L28-N434W antibody, the FH4-N434W antibody, and the 6RL #9-N434W antibody.

[0495] FIG. 23 shows the relationship of a designed amino acid distribution (indicated as Design) to the amino acid distribution (indicated as Library) for the sequence information on 132 clones isolated from E. coli introduced with a gene library of antibodies that bind to antigens in a pH-dependent manner. The horizontal axis indicates amino acid positions in the Kabat numbering system. The vertical axis indicates % amino acid distribution.

[0496] FIG. 24 shows sensorgrams for anti-IL-6R antibody (tocilizumab), antibody 6RpH #01, antibody 6RpH #02, and antibody 6RpH #03 at pH 7.4. The horizontal axis shows time, and the vertical axis shows RU value.

[0497] FIG. 25 shows sensorgrams for anti-IL-6R antibody (tocilizumab), antibody 6RpH #01, antibody 6RpH #02, and antibody 6RpH #03 at pH 6.0. The horizontal axis shows time, and the vertical axis shows RU value.

[0498] FIG. 26 shows changes in the average plasma concentration of hsIL-6R in the non-antibody administration group, and the Fv4-mIgG1, Fv4-mIgG2a, Fv4-mF3, Fv4-mFa30, and H54 / L28-mF3 administration groups.

[0499] FIG. 27 shows changes in the antibody titer of mouse anti-human IL-6 receptor antibody (anti-hsIL-6R antibody) for each individual in the Fv4-mIgG1 administration group.

[0500] FIG. 28 shows changes in the antibody titer of mouse anti-human IL-6 receptor antibody (anti-hsIL-6R antibody) for each individual in the Fv4-mIgG2a administration group.

[0501] FIG. 29 shows changes in the antibody titer of mouse anti-human IL-6 receptor antibody (anti-hsIL-6R antibody) for each individual in the Fv4-mF3 administration group.

[0502] FIG. 30 shows changes in the antibody titer of mouse anti-human IL-6 receptor antibody (anti-hsIL-6R antibody) for each individual in the Fv4-mFa30 administration group.

[0503] FIG. 31 shows changes in the antibody titer of mouse anti-human IL-6 receptor antibody (anti-hsIL-6R antibody) for each individual in the H54 / L28-mF3 administration group.

[0504] FIG. 32A is a schematic diagram showing the relationship between the genomic DNA structure of the mouse interleukin-6 receptor (Il6ra) gene (1) and the knock-in vector to be inserted (2). The knock-in vector has the full-length human interleukin-6-receptor (hIL6R) cDNA, the hp7 sequence, a poly-A addition signal, and a neomycin-resistance gene.

[0505] FIG. 32B is a schematic diagram showing how the genomic DNA of the mouse interleukin-6 receptor gene (a) and the knock-in vector (b) undergo homologous recombination to form a knock-in genomic DNA (c). Furthermore, it shows the process of completing the human interleukin-6 receptor gene knock-in allele (d) by allowing Cre recombinase to act on (c) to remove the neomycin-resistance gene cassette. The arrows in the figure indicate the positions for setting primers used for detecting the knocked-in human interleukin-6 receptor gene.

[0506] FIG. 33 shows a representative example of PCR which analyzed each genotype obtained in the process of establishing the human interleukin-6 receptor gene knock-in mice.

[0507] FIG. 34 shows the expression profile of the interleukin-6 receptor gene in the wild-type mouse and the human interleukin-6 receptor gene knock-in mouse.

[0508] FIG. 35 is a graph showing the results of measuring the plasma concentration of soluble human interleukin-6 receptor (hsIL-6R) in wild-type mice and homozygous and heterozygous human interleukin-6 receptor gene knock-in mice. KI / KI, KI / +, and + / + indicate the homozygous knock-in mice, heterozygous knock-in mice, and the wild-type, respectively.

[0509] FIG. 36 is a graph showing the species-specific reactivity to interleukin-6 (ligand) in the wild-type mice and homozygous human interleukin-6 receptor gene knock-in mice.MODE FOR CARRYING OUT THE INVENTION

[0510] The definitions and detailed description below are provided to help the understanding of the present invention illustrated herein.Amino Acids

[0511] Herein, amino acids are described in one- or three-letter codes or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.Antigen

[0512] Herein, an antigen is not particularly limited, and may be any antigen as long as it is a molecule that may induce the immune system of an organism and thereby become a target of the immune response in that organism. Preferred examples of such antigens include molecules that are expressed specifically in tumor cells but not expressed in normal cells (neoepitopes). Molecules that are expressed in foreign biological species such as bacteria and viruses that infect an organism but not expressed in the organism are also preferred. The phrase “expressed specifically in tumor cells but not expressed in normal cells” or “expressed in foreign biological species that infect an organism but not expressed in the organism” means that there is a qualitative or quantitative difference in the molecule between “tumor cells and normal cells” or “foreign biological species that infect an organism and the organism”. For example, even if a molecule is expressed in normal cells, if the amount of the molecule expressed in tumor cells is far greater than the amount expressed in the normal cells, it can be said in the present invention that there is a quantitative difference in the molecule between tumor cells and normal cells. Furthermore, even if the expression level of a polypeptide consisting of the same amino acid sequence is similar in tumor cells and normal cells, if the expressed polypeptide has undergone posttranslational modification such as phosphorylation in tumor cells but not in normal cells, it can be said in the present invention that there is a qualitative difference in the molecule between tumor cells and normal cells.

[0513] For such molecules, preferred tumor antigens may include: ALK receptor (pleiotrophin receptor), pleiotrophin; KS 1 / 4 pancreas carcinoma antigen; ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostate specific antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globule antigen; colorectal tumor-associated antigens such as CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA; Burkitt's lymphoma antigen-38.13; CD19; human B-lymphoma antigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specific transplantation-type cell-surface antigen (TSTA); virally-induced tumor antigens including T-antigen, DNA tumor viruses and Envelope antigens of RNA tumor viruses; oncofetal antigens such as CEA of colon, 5T4 oncofetal trophoblast glycoprotein, and bladder tumor oncofetal antigen; alpha-fetoprotein; differentiation antigens such as human lung carcinoma antigens L6 and L20; antigens of fibrosarcoma; human leukemia T cell antigen-Gp37; neoglycoprotein; sphingolipids; breast cancer antigens such as EGFR (epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphic epithelial mucin (PEM) antigen; malignant human lymphocyte antigen-APO-1; differentiation antigens such as I antigen found in fetal erythrocytes; primary endoderm I antigen found in adult erythrocytes; preimplantation embryos; I(Ma) found in gastric cancer; M18, M39 found in mammary epithelium; SSEA-1 found in myeloid cells; VEP8; VEP9; Myl; VIM-D5; D156-22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovarian cancer; C14 found in colon cancer; F3 found in lung cancer; AH6 found in gastric cancer; Y hapten; Ley found in embryonal carcinoma cells; TL5 (blood group A); EGF receptor found in A431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2 found in embryonal carcinoma cells; gastric cancer antigen; CO-514 (blood group Lea) found in adenocarcinomas; NS-10 found in adenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor of A431 cells; MH2 (blood group ALeb / Ley) found in colon cancer; 19.9 found in colon cancer; gastric cancer mucins; T5A7 found in myeloid cells; R24 found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos; subcutaneous T cell lymphoma antigen; MART-1 antigen; sialyl Tn (STn) antigen; colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigen ART1; paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen); Neuro-oncological ventral antigen 2 (NOVA2); hemocyte carcinoma antigen gene 520; tumor-associated antigen CO-029; tumor-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B 1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1); YKL-40, and fragments of any of the aforementioned polypeptides or structures produced by modifying them (for example, modified phosphate group or sugar chain of the above-mentioned).

[0514] Antigens of foreign biological species include molecules expressed in: Bacillus anthraces, Clostridium botulinum, Yersinia pestis, Variola major (smallpox) and other poxviruses, Francisella tularensis (tularemia), and those that cause viral hemorrhagic fever, Arenaviruses such as LCM, Junin virus, Machupo virus, Guanarito virus, and those that cause Lassa fever, Bunyaviruses and Hantaviruses such as those that cause Rift-valley fever, Calicivirus, hepatitis A, hepatitis B, hepatitis C, viral encephalitis such as West Nile Virus, LaCrosse, California encephalitis, VEE, EEE, WEE, and Japanese encephalitis virus, Kyasanur forest virus, tickborne hemorrhagic fever virus, Crimean-Congo hemorrhagic fever virus, tickborne encephalitis viruses, Yellow fever, multidrug-resistant TB, influenza, other rickettsiae and rabies, Flavirus, Dengue, Filovirus, Ebola, Marburg Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), ricin toxin (derived from Ricinus communis), epsilon toxin of Clostridium perfringens, Staphylococcus enterotoxin B, Typhus fever (Rickettsia prowazekii), food and water-borne pathogens, bacteria such as diarrheagenic E. coli, pathogenic Vibrios, Shigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, and Yersinia enterocolitica; and protozoas such as Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma, and Microsporidia.

[0515] Other antigens include, for example, the molecules below: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAMS, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (Decay accelerating factor), des (1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV MB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, TAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha4 / beta7, integrin alpha5 (alpha V), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Mullerian-inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, 5100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (for example, T-cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG; TSLP, tumor associated antigen CA125, tumor associated antigen expressing Lewis-Y associated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, SIP, Acetylcholine receptor, AdipoR1, AdipoR2, ADP ribosyl cyclase-1, alpha-4 / beta-7 integrin, alpha-5 / beta-1 integrin, alpha-v / beta-6 integrin, alphavbeta1 integrin, Angiopoietin ligand-2, Angptl2, Anthrax, Cadherin, Carbonic anhydrase-IX, CD105, CD155, CD158a, CD37, CD49b, CD51, CD70, CD72, Claudin 18, Clostridium difficile toxin, CS1, Delta-like protein ligand 4, DHICA oxidase, Dickkopf-1 ligand, Dipeptidyl peptidase IV, EPOR, F protein of RSV, Factor Ia, FasL, Folate receptor alpha, Glucagon receptor, Glucagon-like peptide 1 receptor, Glutamate carboxypeptidase II, GMCSFR, Hepatitis C virus E2 glycoprotein, Hepcidin, IL-17 receptor, IL-22 receptor, IL-23 receptor, IL-3 receptor, Kit tyrosine kinase, Leucine Rich Alpha-2-Glycoprotein 1 (LRG1), Lysosphingolipid receptor, Membrane glycoprotein OX2, Mesothelin, MET, MICA, MUC-16, Myelin associated glycoprotein, Neuropilin-1, Neuropilin-2, Nogo receptor, PLXNA1, PLXNA2, PLXNA3, PLXNA4A, PLXNA4B, PLXNB1, PLXNB2, PLXNB3, PLXNC1, PLXND1, Programmed cell death ligand 1, Proprotein convertase PC9, P-selectin glycoprotein ligand-1, RAGE, Reticulon 4, RF, RON-8, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, SEMA7A, Shiga like toxin II, Sphingosine-1-phosphate receptor-1, ST2, Staphylococcal lipoteichoic acid, Tenascin, TG2, Thymic stromal lymphoprotein receptor, TNF superfamily receptor 12A, Transmembrane glycoprotein NMB, TREM-1, TREM-2, Trophoblast glycoprotein, TSH receptor, TTR, Tubulin, and ULBP2; and receptors for hormone and growth factors.Epitope

[0516] “Epitope” means an antigenic determinant in an antigen, and refers to an antigen site to which the antigen-binding domain of an antigen-binding molecule disclosed herein binds. Thus, for example, the epitope can be defined according to its structure. Alternatively, the epitope may be defined according to the antigen-binding activity of an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues forming the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be specified by its specific sugar chain structure.

[0517] A linear epitope is an epitope that contains an epitope whose primary amino acid sequence is recognized. Such a linear epitope typically contains at least three and most commonly at least five, for example, about 8 to 10 or 6 to 20 amino acids in its specific sequence.

[0518] In contrast to the linear epitope, “conformational epitope” is an epitope in which the primary amino acid sequence containing the epitope is not the only determinant of the recognized epitope (for example, the primary amino acid sequence of a conformational epitope is not necessarily recognized by an epitope-defining antibody). Conformational epitopes may contain a greater number of amino acids compared to linear epitopes. A conformational epitope-recognizing antibody recognizes the three-dimensional structure of a peptide or protein.

[0519] For example, when a protein molecule folds and forms a three-dimensional structure, amino acids and / or polypeptide main chains that form a conformational epitope become aligned, and the epitope is made recognizable by the antibody. Methods for determining epitope conformations include, for example, X ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance, but are not limited thereto. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).Binding Activity

[0520] Examples of a method for assessing the epitope binding by a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain are described below. According to the examples below, methods for assessing the epitope binding by a test antigen-binding molecule containing an antigen-binding domain for an antigen other than IL-6 receptor, can also be appropriately conducted.

[0521] For example, whether a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain recognizes a linear epitope in the IL-6 receptor molecule can be confirmed for example as mentioned below. A linear peptide comprising an amino acid sequence forming the extracellular domain of IL-6 receptor is synthesized for the above purpose. The peptide can be synthesized chemically, or obtained by genetic engineering techniques using a region encoding the amino acid sequence corresponding to the extracellular domain in an IL-6 receptor cDNA. Then, a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain is assessed for its binding activity towards a linear peptide comprising the amino acid sequence forming the extracellular domain. For example, an immobilized linear peptide can be used as an antigen by ELISA to evaluate the binding activity of the antigen-binding molecule towards the peptide. Alternatively, the binding activity towards a linear peptide can be assessed based on the level that the linear peptide inhibits the binding of the antigen-binding molecule to IL-6 receptor-expressing cells. These tests can demonstrate the binding activity of the antigen-binding molecule towards the linear peptide.

[0522] Whether a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain recognizes a conformational epitope can be assessed as follows. IL-6 receptor-expressing cells are prepared for the above purpose. A test antigen-binding molecule containing an IL-6 receptor antigen-binding domain can be determined to recognize a conformational epitope when it strongly binds to IL-6 receptor-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide comprising an amino acid sequence forming the extracellular domain of IL-6 receptor. Herein, “not substantially bind” means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less compared to the binding activity towards cells expressing human IL-6 receptor.

[0523] Methods for assaying the binding activity of a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain towards IL-6 receptor-expressing cells include, for example, the methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the assessment can be performed based on the principle of ELISA or fluorescence activated cell sorting (FACS) using IL-6 receptor-expressing cells as antigen.

[0524] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain towards IL-6 receptor-expressing cells can be assessed quantitatively by comparing the levels of signal generated by enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate onto which IL-6 receptor-expressing cells are immobilized. Then, the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, when FACS is used, a dilution series of a test antigen-binding molecule is prepared, and the antibody binding titer for IL-6 receptor-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule towards IL-6 receptor-expressing cells.

[0525] The binding of a test antigen-binding molecule towards an antigen expressed on the surface of cells suspended in buffer or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices:

[0526] FACSCanto™ II

[0527] FACSAria™

[0528] FACSArray™

[0529] FACSVantage™ SE

[0530] FACSCalibur™ (all are trade names of BD Biosciences)

[0531] EPICS ALTRA HyPerSort

[0532] Cytomics FC 500

[0533] EPICS XL-MCL ADC EPICS XL ADC

[0534] Cell Lab Quanta / Cell Lab Quanta SC (all are trade names of Beckman Coulter).

[0535] Preferable methods for assaying the binding activity of a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain towards an antigen include, for example, the following method. First, IL-6 receptor-expressing cells are reacted with a test antigen-binding molecule, and then this is stained with an FITC-labeled secondary antibody that recognizes the antigen-binding molecule. The test antigen-binding molecule is appropriately diluted with a suitable buffer to prepare the molecule at a desired concentration. For example, the molecule can be used at a concentration within the range of 10 μg / ml to 10 ng / ml. Then, the fluorescence intensity and cell count are determined using FACSCalibur (BD). The fluorescence intensity obtained by analysis using the CELL QUEST Software (BD), i.e., the Geometric Mean value, reflects the quantity of antibody bound to cells. That is, the binding activity of a test antigen-binding molecule, which is represented by the quantity of the test antigen-binding molecule bound, can be determined by measuring the Geometric Mean value.

[0536] Whether a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain shares a common epitope with another antigen-binding molecule can be assessed based on the competition between the two molecules for the same epitope. The competition between antigen-binding molecules can be detected by cross-blocking assay or the like. For example, the competitive ELISA assay is a preferred cross-blocking assay.

[0537] Specifically, in cross-blocking assay, the IL-6 receptor protein immobilized to the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competitor antigen-binding molecule, and then a test antigen-binding molecule is added thereto. The quantity of test antigen-binding molecule bound to the IL-6 receptor protein in the wells is indirectly correlated with the binding ability of a candidate competitor antigen-binding molecule that competes for the binding to the same epitope. That is, the greater the affinity of the competitor antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule towards the IL-6 receptor protein-coated wells.

[0538] The quantity of the test antigen-binding molecule bound to the wells via the IL-6 receptor protein can be readily determined by labeling the antigen-binding molecule in advance. For example, a biotin-labeled antigen-binding molecule is measured using an avidin / peroxidase conjugate and appropriate substrate. In particular, cross-blocking assay that uses enzyme labels such as peroxidase is called “competitive ELISA assay”. The antigen-binding molecule can also be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabels, fluorescent labels, and such are known.

[0539] When the candidate competitor antigen-binding molecule can block the binding by a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain by at least 20%, preferably at least 20 to 50%, and more preferably at least 50% compared to the binding activity in a control experiment conducted in the absence of the competitor antigen-binding molecule, the test antigen-binding molecule is determined to substantially bind to the same epitope bound by the competitor antigen-binding molecule, or compete for the binding to the same epitope.

[0540] When the structure of an epitope bound by a test antigen-binding molecule containing an IL-6 receptor antigen-binding domain has already been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activities of the two antigen-binding molecules towards a peptide prepared by introducing amino acid mutations into the peptide forming the epitope.

[0541] To measure the above binding activities, for example, the binding activities of test and control antigen-binding molecules towards a linear peptide into which a mutation is introduced are compared in the above ELISA format. Besides the ELISA methods, the binding activity towards the mutant peptide bound to a column can be determined by flowing test and control antigen-binding molecules in the column, and then quantifying the antigen-binding molecule eluted in the elution solution. Methods for adsorbing a mutant peptide to a column, for example, in the form of a GST fusion peptide, are known.

[0542] Alternatively, when the identified epitope is a conformational epitope, whether test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, IL-6 receptor-expressing cells and cells expressing IL-6 receptor with a mutation introduced into the epitope are prepared. The test and control antigen-binding molecules are added to a cell suspension prepared by suspending these cells in an appropriate buffer such as PBS. Then, the cell suspensions are appropriately washed with a buffer, and an FITC-labeled antibody that recognizes the test and control antigen-binding molecules is added thereto. The fluorescence intensity and number of cells stained with the labeled antibody are determined using FACSCalibur (BD). The test and control antigen-binding molecules are appropriately diluted using a suitable buffer, and used at desired concentrations. For example, they may be used at a concentration within the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity determined by analysis using the CELL QUEST Software (BD), i.e., the Geometric Mean value, reflects the quantity of labeled antibody bound to cells. That is, the binding activities of the test and control antigen-binding molecules, which are represented by the quantity of labeled antibody bound, can be determined by measuring the Geometric Mean value.

[0543] In the above method, whether an antigen-binding molecule does “not substantially bind to cells expressing mutant IL-6 receptor” can be assessed, for example, by the following method. First, the test and control antigen-binding molecules bound to cells expressing mutant IL-6 receptor are stained with a labeled antibody. Then, the fluorescence intensity of the cells is determined. When FACSCalibur is used for fluorescence detection by flow cytometry, the determined fluorescence intensity can be analyzed using the CELL QUEST Software. From the Geometric Mean values in the presence and absence of the polypeptide complex, the comparison value (ΔGeo-Mean) can be calculated according to the following formula to determine the ratio of increase in fluorescence intensity as a result of the binding by the antigen-binding molecule.ΔGeo-Mean=Geo-Mean (in the presence of the polypeptide complex) / Geo-Mean (in the absence of the polypeptide complex)

[0544] The Geometric Mean comparison value (ΔGeo-Mean value for the mutant IL-6 receptor molecule) determined by the above analysis, which reflects the quantity of a test antigen-binding molecule bound to cells expressing mutant IL-6 receptor, is compared to the ΔGeo-Mean comparison value that reflects the quantity of the test antigen-binding molecule bound to IL-6 receptor-expressing cells. In this case, the concentrations of the test antigen-binding molecule used to determine the ΔGeo-Mean comparison values for IL-6 receptor-expressing cells and cells expressing mutant IL-6 receptor are particularly preferably adjusted to be equal or substantially equal. An antigen-binding molecule that has been confirmed to recognize an epitope in IL-6 receptor is used as a control antigen-binding molecule.

[0545] If the ΔGeo-Mean comparison value of a test antigen-binding molecule for cells expressing mutant IL-6 receptor is smaller than the ΔGeo-Mean comparison value of the test antigen-binding molecule for IL-6 receptor-expressing cells by at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15%, then the test antigen-binding molecule “does not substantially bind to cells expressing mutant IL-6 receptor”. The formula for determining the Geo-Mean (Geometric Mean) value is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison shows that the comparison values are substantially equivalent, the epitope for the test and control antigen-binding molecules can be determined to be the same.Antigen-Binding Domain

[0546] An “antigen-binding domain” may be of any structure as long as it binds to an antigen of interest. Such domains preferably include, for example:

[0547] antibody heavy-chain and light-chain variable regions;

[0548] a module of about 35 amino acids called A domain which is contained in the in vivo cell membrane protein Avimer (WO 2004 / 044011, WO 2005 / 040229);

[0549] Adnectin containing the 10Fn3 domain which binds to the protein moiety of fibronectin, a glycoprotein expressed on cell membrane (WO 2002 / 032925);

[0550] Affibody which is composed of a 58-amino acid three-helix bundle based on the scaffold of the IgG-binding domain of Protein A (WO 1995 / 001937);

[0551] Designed Ankyrin Repeat proteins (DARPins) which are a region exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a subunit consisting of a turn comprising 33 amino acid residues, two antiparallel helices, and a loop is repeatedly stacked (WO 2002 / 020565);

[0552] Anticalins and such, which are domains consisting of four loops that support one side of a barrel structure composed of eight circularly arranged antiparallel strands that are highly conserved among lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO 2003 / 029462); and

[0553] the concave region formed by the parallel-sheet structure inside the horseshoe-shaped structure constituted by stacked repeats of the leucine-rich-repeat (LRR) module of the variable lymphocyte receptor (VLR) which does not have the immunoglobulin structure and is used in the system of acquired immunity in jawless vertebrate such as lampery and hagfish (WO 2008 / 016854). Preferred antigen-binding domains of the present invention include, for example, those having antibody heavy-chain and light-chain variable regions. Preferred examples of antigen-binding domains include “single chain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv 2 (scFv2)”, “Fab”, and “F(ab′)2”.

[0554] The antigen-binding domains of antigen-binding molecules of the present invention can bind to an identical epitope. Such epitope can be present, for example, in a protein comprising the amino acid sequence of SEQ ID NO: 1. Alternatively, each of the antigen-binding domains of antigen-binding molecules of the present invention can bind to a different epitope. Herein, the different epitope can be present in, for example, a protein comprising the amino acid sequence of SEQ ID NO: 1.Specificity

[0555] “Specific” means that one of molecules that specifically binds to does not show any significant binding to molecules other than a single or a number of binding partner molecules. Furthermore, “specific” is also used when an antigen-binding domain is specific to a particular epitope among multiple epitopes in an antigen. When an epitope bound by an antigen-binding domain is contained in multiple different antigens, antigen-binding molecules containing the antigen-binding domain can bind to various antigens that have the epitope.Antibodies

[0556] Herein, “antibody” refers to a natural immunoglobulin or an immunoglobulin produced by partial or complete synthesis. Antibodies can be isolated from natural sources such as naturally-occurring plasma and serum, or culture supernatants of antibody-producing hybridomas. Alternatively, antibodies can be partially or completely synthesized using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of an immunoglobulin isotype or subclass belonging thereto. Known human immunoglobulins include antibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the present invention include IgG1, IgG2, IgG3, and IgG4.

[0557] Methods for producing an antibody with desired binding activity are known to those skilled in the art. Below is an example that describes a method for producing an antibody that binds to IL-6 receptor (anti-IL-6 receptor antibody). Antibodies that bind to an antigen other than IL-6 receptor can also be produced according to the example described below.

[0558] Anti-IL-6 receptor antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. The anti-IL-6 receptor antibodies preferably produced are monoclonal antibodies derived from mammals. Such mammal-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector carrying an antibody gene by genetic engineering techniques. “Humanized antibodies” or “chimeric antibodies” are included in the monoclonal antibodies of the present invention.

[0559] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as described below. Specifically, mammals are immunized by conventional immunization methods using an IL-6 receptor protein as a sensitizing antigen. Resulting immune cells are fused with known parental cells by conventional cell fusion methods. Then, hybridomas producing an anti-IL-6 receptor antibody can be selected by screening for monoclonal antibody-producing cells using conventional screening methods.

[0560] Specifically, monoclonal antibodies are prepared as mentioned below. First, the IL-6 receptor gene whose nucleotide sequence is disclosed in SEQ ID NO: 2 can be expressed to produce an IL-6 receptor protein shown in SEQ ID NO: 1, which will be used as a sensitizing antigen for antibody preparation. That is, a gene sequence encoding IL-6 receptor is inserted into a known expression vector, and appropriate host cells are transformed with this vector. The desired human IL-6 receptor protein is purified from the host cells or their culture supernatants by known methods. In order to obtain soluble IL-6 receptor from culture supernatants, for example, a protein consisting of the amino acids at positions 1 to 357 in the IL-6 receptor polypeptide sequence of SEQ ID NO: 1, such as described in Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968), is expressed as a soluble IL-6 receptor, instead of the IL-6 receptor protein of SEQ ID NO: 1. Purified natural IL-6 receptor protein can also be used as a sensitizing antigen.

[0561] The purified IL-6 receptor protein can be used as a sensitizing antigen for immunization of mammals. A partial IL-6 receptor peptide may also be used as a sensitizing antigen. In this case, a partial peptide can be prepared by chemical synthesis based on the amino acid sequence of human IL-6 receptor, or by inserting a partial IL-6 receptor gene into an expression vector for expression. Alternatively, a partial peptide can be produced by degrading an IL-6 receptor protein with a protease. The length and region of the partial IL-6 receptor peptide are not limited to particular embodiments. A preferred region can be arbitrarily selected from the amino acid sequence at amino acid positions 20 to 357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids forming a peptide to be used as a sensitizing antigen is preferably at least five or more, six or more, or seven or more. More specifically, a peptide of 8 to 50 residues, more preferably 10 to 30 residues can be used as a sensitizing antigen.

[0562] For sensitizing antigen, alternatively it is possible to use a fusion protein prepared by fusing a desired partial polypeptide or peptide of the IL-6 receptor protein with a different polypeptide. For example, antibody Fc fragments and peptide tags are preferably used to produce fusion proteins to be used as sensitizing antigens. Vectors for expression of such fusion proteins can be constructed by fusing in frame genes encoding two or more desired polypeptide fragments and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. Press). Methods for preparing IL-6 receptor to be used as a sensitizing antigen, and immunization methods using IL-6 receptor are specifically described in WO 2003 / 000883, WO 2004 / 022754, WO 2006 / 006693, and such.

[0563] There is no particular limitation on the mammals to be immunized with the sensitizing antigen. However, it is preferable to select the mammals by considering their compatibility with the parent cells to be used for cell fusion. In general, rodents such as mice, rats, and hamsters, rabbits, and monkeys are preferably used.

[0564] The above animals are immunized with a sensitizing antigen by known methods. Generally performed immunization methods include, for example, intraperitoneal or subcutaneous injection of a sensitizing antigen into mammals. Specifically, a sensitizing antigen is appropriately diluted with PBS (Phosphate-Buffered Saline), physiological saline, or the like. If desired, a conventional adjuvant such as Freund's complete adjuvant is mixed with the antigen, and the mixture is emulsified. Then, the sensitizing antigen is administered to a mammal several times at 4- to 21-day intervals. Appropriate carriers may be used in immunization with the sensitizing antigen. In particular, when a low-molecular-weight partial peptide is used as the sensitizing antigen, it is sometimes desirable to couple the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

[0565] Alternatively, hybridomas producing a desired antibody can be prepared using DNA immunization as mentioned below. DNA immunization is an immunization method that confers immunostimulation by expressing a sensitizing antigen in an animal immunized as a result of administering a vector DNA constructed to allow expression of an antigen protein-encoding gene in the animal. As compared to conventional immunization methods in which a protein antigen is administered to animals to be immunized, DNA immunization is expected to be superior in that:

[0566] immunostimulation can be provided while retaining the structure of a membrane protein such as IL-6 receptor; and

[0567] there is no need to purify the antigen for immunization.

[0568] In order to prepare a monoclonal antibody of the present invention using DNA immunization, first, a DNA expressing an IL-6 receptor protein is administered to an animal to be immunized. The IL-6 receptor-encoding DNA can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector, and then this is administered to an animal to be immunized. Preferably used expression vectors include, for example, commercially-available expression vectors such as pcDNA3.1. Vectors can be administered to an organism using conventional methods. For example, DNA immunization is performed by using a gene gun to introduce expression vector-coated gold particles into cells in the body of an animal to be immunized. Antibodies that recognized IL-6 receptor can also be produced by the methods described in WO 2003 / 104453.

[0569] After immunizing a mammal as described above, an increase in the titer of an IL-6 receptor-binding antibody is confirmed in the serum. Then, immune cells are collected from the mammal, and then subjected to cell fusion. In particular, splenocytes are preferably used as immune cells.

[0570] A mammalian myeloma cell is used as a cell to be fused with the above-mentioned immune cells. The myeloma cells preferably comprise a suitable selection marker for screening. A selection marker confers characteristics to cells for their survival (or death) under a specific culture condition. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selection markers. Cells with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in a HAT selection medium, and are thus killed. However, when the cells are fused with normal cells, they can continue DNA synthesis using the salvage pathway of the normal cells, and therefore they can grow even in the HAT selection medium.

[0571] HGPRT-deficient and TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5′-bromodeoxyuridine, respectively. Normal cells are killed because they incorporate these pyrimidine analogs into their DNA. Meanwhile, cells that are deficient in these enzymes can survive in the selection medium, since they cannot incorporate these pyrimidine analogs. In addition, a selection marker referred to as G418 resistance provided by the neomycin-resistant gene confers resistance to 2-deoxystreptamine antibiotics (gentamycin analogs). Various types of myeloma cells that are suitable for cell fusion are known.

[0572] For example, myeloma cells including the following cells can be preferably used:

[0573] P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);

[0574] P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7);

[0575] NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519);

[0576] MPC-11 (Cell (1976) 8 (3), 405-415);

[0577] SP2 / 0 (Nature (1978) 276 (5685), 269-270);

[0578] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);

[0579] S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

[0580] R210 (Nature (1979) 277 (5692), 131-133), etc.

[0581] Cell fusions between the immunocytes and myeloma cells are essentially carried out using known methods, for example, a method by Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46).

[0582] More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion-promoting agent. The fusion-promoting agents include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If required, an auxiliary substance such as dimethyl sulfoxide is also added to improve fusion efficiency.

[0583] The ratio of immune cells to myeloma cells may be determined at one's own discretion, preferably, for example, one myeloma cell for every one to ten immunocytes. Culture media to be used for cell fusions include, for example, media that are suitable for the growth of myeloma cell lines, such as RPMI1640 medium and MEM medium, and other conventional culture medium used for this type of cell culture. In addition, serum supplements such as fetal calf serum (FCS) may be preferably added to the culture medium.

[0584] For cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed well in the above culture medium. Then, a PEG solution (for example, the average molecular weight is about 1,000 to 6,000) prewarmed to about 37° C. is added thereto at a concentration of generally 30% to 60% (w / v). This is gently mixed to produce desired fusion cells (hybridomas). Then, an appropriate culture medium mentioned above is gradually added to the cells, and this is repeatedly centrifuged to remove the supernatant. Thus, cell fusion agents and such which are unfavorable to hybridoma growth can be removed.

[0585] The hybridomas thus obtained can be selected by culture using a conventional selective medium, for example, HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culture in the above HAT medium for a sufficient period of time. Typically, the period is several days to several weeks. Then, hybridomas producing the desired antibody are screened and singly cloned by conventional limiting dilution methods.

[0586] The hybridomas thus obtained can be selected using a selection medium based on the selection marker possessed by the myeloma used for cell fusion. For example, HGPRT- or TK-deficient cells can be selected by culture using the HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively proliferate in the HAT medium. Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culture in the above HAT medium for a sufficient period of time. Specifically, desired hybridomas can be selected by culture for generally several days to several weeks. Then, hybridomas producing the desired antibody are screened and singly cloned by conventional limiting dilution methods.

[0587] Desired antibodies can be preferably selected and singly cloned by screening methods based on known antigen / antibody reaction. For example, an IL-6 receptor-binding monoclonal antibody can bind to IL-6 receptor expressed on the cell surface. Such a monoclonal antibody can be screened by fluorescence activated cell sorting (FACS). FACS is a system that assesses the binding of an antibody to cell surface by analyzing cells contacted with a fluorescent antibody using laser beam, and measuring the fluorescence emitted from individual cells.

[0588] To screen for hybridomas that produce a monoclonal antibody of the present invention by FACS, IL-6 receptor-expressing cells are first prepared. Cells preferably used for screening are mammalian cells in which IL-6 receptor is forcedly expressed. As control, the activity of an antibody to bind to cell-surface IL-6 receptor can be selectively detected using non-transformed mammalian cells as host cells. Specifically, hybridomas producing an anti-IL-6 receptor monoclonal antibody can be isolated by selecting hybridomas that produce an antibody which binds to cells forced to express IL-6 receptor, but not to host cells.

[0589] Alternatively, the activity of an antibody to bind to immobilized IL-6 receptor-expressing cells can be assessed based on the principle of ELISA. For example, IL-6 receptor-expressing cells are immobilized to the wells of an ELISA plate. Culture supernatants of hybridomas are contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. When the monoclonal antibodies are derived from mouse, antibodies bound to the cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas producing a desired antibody having the antigen-binding ability are selected by the above screening, and they can be cloned by a limiting dilution method or the like.

[0590] Monoclonal antibody-producing hybridomas thus prepared can be passaged in a conventional culture medium, and stored in liquid nitrogen for a long period.

[0591] The above hybridomas are cultured by a conventional method, and desired monoclonal antibodies can be prepared from the culture supernatants. Alternatively, the hybridomas are administered to and grown in compatible mammals, and monoclonal antibodies are prepared from the ascites. The former method is suitable for preparing antibodies with high purity.

[0592] Antibodies encoded by antibody genes that are cloned from antibody-producing cells such as the above hybridomas can also be preferably used. A cloned antibody gene is inserted into an appropriate vector, and this is introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting the genes into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for producing recombinant antibodies are also known as described below.

[0593] For example, a cDNA encoding the variable region (V region) of an anti-IL-6 receptor antibody is prepared from hybridoma cells expressing the anti-IL-6 receptor antibody. For this purpose, total RNA is first extracted from hybridomas. Methods used for extracting mRNAs from cells include, for example:

[0594] the guanidine ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299), and

[0595] the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

[0596] Extracted mRNAs can be purified using the mRNA Purification Kit (GE Healthcare Bioscience) or such. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bioscience), are also commercially available. mRNAs can be prepared from hybridomas using such kits. cDNAs encoding the antibody V region can be synthesized from the prepared mRNAs using a reverse transcriptase.

[0597] cDNAs can be synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) or such. Furthermore, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5′-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932) can be appropriately used to synthesize and amplify cDNAs. In such a cDNA synthesis process, appropriate restriction enzyme sites described below may be introduced into both ends of a cDNA.

[0598] The cDNA fragment of interest is purified from the resulting PCR product, and then this is ligated to a vector DNA. A recombinant vector is thus constructed, and introduced into E. coli or such. After colony selection, the desired recombinant vector can be prepared from the colony-forming E. coli. Then, whether the recombinant vector has the cDNA nucleotide sequence of interest is tested by a known method such as the dideoxy nucleotide chain termination method.

[0599] The 5′-RACE method which uses primers to amplify the variable region gene is conveniently used for isolating the gene encoding the variable region. First, a 5′-RACE cDNA library is constructed by cDNA synthesis using RNAs extracted from hybridoma cells as a template. A commercially available kit such as the SMART RACE cDNA amplification kit is appropriately used to synthesize the 5′-RACE cDNA library.

[0600] The antibody gene is amplified by PCR using the prepared 5′-RACE cDNA library as a template. Primers for amplifying the mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Therefore, it is preferable that the subclass is determined in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

[0601] Specifically, for example, primers that allow amplification of genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains are used to isolate mouse IgG-encoding genes. In general, a primer that anneals to a constant region site close to the variable region is used as a 3′-side primer to amplify an IgG variable region gene. Meanwhile, a primer attached to a 5′ RACE cDNA library construction kit is used as a 5′-side primer.

[0602] PCR products thus amplified are used to reshape immunoglobulins composed of a combination of heavy and light chains. A desired antibody can be selected using the IL-6 receptor-binding activity of a reshaped immunoglobulin as an indicator. For example, when the objective is to isolate an antibody against IL-6 receptor, it is more preferred that the binding of the antibody to IL-6 receptor is specific. An IL-6 receptor-binding antibody can be screened, for example, by the following steps:

[0603] (1) contacting an IL-6 receptor-expressing cell with an antibody comprising the V region encoded by a cDNA isolated from a hybridoma;

[0604] (2) detecting the binding of the antibody to the IL-6 receptor-expressing cell; and

[0605] (3) selecting an antibody that binds to the IL-6 receptor-expressing cell.

[0606] Methods for detecting the binding of an antibody to IL-6 receptor-expressing cells are known. Specifically, the binding of an antibody to IL-6 receptor-expressing cells can be detected by the above-described techniques such as FACS. Immobilized samples of IL-6 receptor-expressing cells are appropriately used to assess the binding activity of an antibody.

[0607] Preferred antibody screening methods that use the binding activity as an indicator also include panning methods using phage vectors. Screening methods using phage vectors are advantageous when the antibody genes are isolated from heavy-chain and light-chain subclass libraries from a polyclonal antibody-expressing cell population. Genes encoding the heavy-chain and light-chain variable regions can be linked by an appropriate linker sequence to form a single-chain Fv (scFv). Phages presenting scFv on their surface can be produced by inserting a gene encoding scFv into a phage vector. The phages are contacted with an antigen of interest. Then, a DNA encoding scFv having the binding activity of interest can be isolated by collecting phages bound to the antigen. This process can be repeated as necessary to enrich scFv having the binding activity of interest.

[0608] After isolation of the cDNA encoding the V region of the anti-IL-6 receptor antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites introduced into both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs in the nucleotide sequence of the antibody gene at a low frequency. Furthermore, a restriction site for an enzyme that produces a sticky end is preferably introduced into a vector to insert a single-copy digested fragment in the correct orientation. The cDNA encoding the V region of the anti-IL-6 receptor antibody is digested as described above, and this is inserted into an appropriate expression vector to construct an antibody expression vector. In this case, if a gene encoding the antibody constant region (C region) and a gene encoding the above V region are fused in-frame, a chimeric antibody is obtained. Herein, “chimeric antibody” means that the origin of the constant region is different from that of the variable region. Thus, in addition to mouse / human heterochimeric antibodies, human / human allochimeric antibodies are included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the above V region gene into an expression vector that already has the constant region. Specifically, for example, a recognition sequence for a restriction enzyme that excises the above V region gene can be appropriately placed on the 5′ side of an expression vector carrying a DNA encoding a desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing in frame the two genes digested with the same combination of restriction enzymes.

[0609] To produce an anti-IL-6 receptor monoclonal antibody, antibody genes are inserted into an expression vector so that the genes are expressed under the control of an expression regulatory region. The expression regulatory region for antibody expression includes, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may be attached to the amino terminus so that the expressed antibody is secreted to the outside of cells. In the Examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) are used as a signal sequence. Meanwhile, other appropriate signal sequences may be attached. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the resulting polypeptide is secreted to the outside of cells as a mature polypeptide. Then, appropriate host cells are transformed with the expression vector, and recombinant cells expressing the anti-IL-6 receptor antibody-encoding DNA are obtained.

[0610] DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are separately inserted into different expression vectors to express the antibody gene. An antibody molecule having the H and L chains can be expressed by co-transfecting the same host cell with vectors into which the H-chain and L-chain genes are respectively inserted. Alternatively, host cells can be transformed with a single expression vector into which DNAs encoding the H and L chains are inserted (see WO 1994 / 11523).

[0611] There are various known host cell / expression vector combinations for antibody preparation by introducing isolated antibody genes into appropriate hosts. All of these expression systems are applicable to isolation of the antigen-binding domains of the present invention. Appropriate eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, the animal cells include, for example, the following cells.

[0612] (1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, human embryonic kidney (HEK) 293, or such;

[0613] (2) amphibian cells: Xenopus oocytes, or such; and

[0614] (3) insect cells: sf9, sf21, Tn5, or such.

[0615] In addition, as a plant cell, an antibody gene expression system using cells derived from the Nicotiana genus such as Nicotiana tabacum is known. Callus cultured cells can be appropriately used to transform plant cells.

[0616] Furthermore, the following cells can be used as fungal cells:

[0617] yeasts: the Saccharomyces genus such as Saccharomyces cerevisiae, and the Pichia genus such as Pichia pastoris; and

[0618] filamentous fungi: the Aspergillus genus such as Aspergillus niger.

[0619] Furthermore, antibody gene expression systems that utilize prokaryotic cells are also known. For example, when using bacterial cells, E. coli cells, Bacillus subtilis cells, and such can suitably be utilized in the present invention. Expression vectors carrying the antibody genes of interest are introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be prepared from the culture of transformed cells.

[0620] In addition to the above-described host cells, transgenic animals can also be used to produce a recombinant antibody. That is, the antibody can be obtained from an animal into which the gene encoding the antibody of interest is introduced. For example, the antibody gene can be constructed as a fusion gene by inserting in frame into a gene that encodes a protein produced specifically in milk. Goat (3-casein or such can be used, for example, as the protein secreted in milk. DNA fragments containing the fused gene inserted with the antibody gene is injected into a goat embryo, and then this embryo is introduced into a female goat. Desired antibodies can be obtained as a protein fused with the milk protein from milk produced by the transgenic goat born from the embryo-recipient goat (or progeny thereof). In addition, to increase the volume of milk containing the desired antibody produced by the transgenic goat, hormones can be administered to the transgenic goat as necessary (Ebert, K. M. et al., Bio / Technology (1994) 12 (7), 699-702).

[0621] When a polypeptide complex described herein is administered to human, an antigen-binding domain derived from a genetically recombinant antibody that has been artificially modified to reduce the heterologous antigenicity against human and such, can be appropriately used as the antigen-binding domain of the complex. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced by known methods.

[0622] An antibody variable region used to produce the antigen-binding domain of a polypeptide complex described herein is generally formed by three complementarity-determining regions (CDRs) that are separated by four framework regions (FRs). CDR is a region that substantially determines the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, the FR-forming amino acid sequences often have high identity even among antibodies with different binding specificities. Therefore, generally, the binding specificity of a certain antibody can be introduced to another antibody by CDR grafting.

[0623] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies prepared by grafting the CDR of a non-human animal antibody such as a mouse antibody to a human antibody and such are known. Common genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting a mouse antibody CDR to a human FR. In overlap extension PCR, a nucleotide sequence encoding a mouse antibody CDR to be grafted is added to primers for synthesizing a human antibody FR. Primers are prepared for each of the four FRs. It is generally considered that when grafting a mouse CDR to a human FR, selecting a human FR that has high identity to a mouse FR is advantageous for maintaining the CDR function. That is, it is generally preferable to use a human FR comprising an amino acid sequence which has high identity to the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.

[0624] Nucleotide sequences to be ligated are designed so that they will be connected to each other in frame. Human FRs are individually synthesized using the respective primers. As a result, products in which the mouse CDR-encoding DNA is attached to the individual FR-encoding DNAs are obtained. Nucleotide sequences encoding the mouse CDR of each product are designed so that they overlap with each other. Then, complementary strand synthesis reaction is conducted to anneal the overlapping CDR regions of the products synthesized using a human antibody gene as template. Human FRs are ligated via the mouse CDR sequences by this reaction.

[0625] The full length V region gene, in which three CDRs and four FRs are ultimately ligated, is amplified using primers that anneal to its 5′- or 3′-end, which are added with suitable restriction enzyme recognition sequences. An expression vector for humanized antibody can be produced by inserting the DNA obtained as described above and a DNA that encodes a human antibody C region into an expression vector so that they will ligate in frame. After the recombinant vector is transfected into a host to establish recombinant cells, the recombinant cells are cultured, and the DNA encoding the humanized antibody is expressed to produce the humanized antibody in the cell culture (see, European Patent Publication No. EP 239400 andInternational Patent Publication No. WO 1996 / 002576).

[0626] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody produced as described above, one can suitably select human antibody FRs that allow CDRs to form a favorable antigen-binding site when ligated through the CDRs. Amino acid residues in FRs may be substituted as necessary, so that the CDRs of a reshaped human antibody form an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into FRs by applying the PCR method used for grafting a mouse CDR into a human FR. More specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. Nucleotide sequence mutations are introduced into the FRs synthesized by using such primers. Mutant FR sequences having the desired characteristics can be selected by measuring and evaluating the activity of the amino acid-substituted mutant antibody to bind to the antigen by the above-mentioned method (Cancer Res. (1993) 53: 851-856).

[0627] Alternatively, desired human antibodies can be obtained by immunizing transgenic animals having the entire repertoire of human antibody genes (see WO 1993 / 012227; WO 1992 / 003918; WO 1994 / 002602; WO 1994 / 025585; WO 1996 / 034096; WO 1996 / 033735) by DNA immunization.

[0628] Furthermore, techniques for preparing human antibodies by panning using human antibody libraries are also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on phage surface by the phage display method. Phages expressing an scFv that binds to the antigen can be selected. The DNA sequence encoding the human antibody V region that binds to the antigen can be determined by analyzing the genes of selected phages. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by fusing the V region sequence in frame with the C region sequence of a desired human antibody, and inserting this into an appropriate expression vector. The expression vector is introduced into cells appropriate for expression such as those described above. The human antibody can be produced by expressing the human antibody-encoding gene in the cells. These methods are already known (see WO 1992 / 001047; WO 1992 / 020791; WO 1993 / 006213; WO 1993 / 011236; WO 1993 / 019172; WO 1995 / 001438; WO 1995 / 015388).

[0629] In addition to the techniques described above, techniques of B cell cloning (identification of each antibody-encoding sequence, cloning and its isolation; use in constructing expression vector in order to prepare each antibody (IgG1, IgG2, IgG3, or IgG4 in particular); and such) such as described in Bernasconi et al. (Science (2002) 298: 2199-2202) or in WO 2008 / 081008 can be appropriately used to isolate antibody genes.EU Numbering System and Kabat's Numbering System

[0630] According to the methods used in the present invention, amino acid positions assigned to antibody CDR and FR are specified according to Kabat's numbering (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Herein, when an antigen-binding molecule is an antibody or antigen-binding fragment, variable region amino acids are indicated according to Kabat's numbering system, while constant region amino acids are indicated according to EU numbering system based on Kabat's amino acid positions.Conditions of Ion ConcentrationConditions of Metal Ion Concentration

[0631] In a non-limiting embodiment of the present invention, the ion concentration refers to a metal ion concentration. “Metal ions” refer to ions of group I elements except hydrogen such as alkaline metals and copper group elements, group II elements such as alkaline earth metals and zinc group elements, group III elements except boron, group IV elements except carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become cations. This is referred to as ionization tendency. Metals with strong ionization tendency are deemed to be chemically active.

[0632] In the present invention, preferred metal ions include, for example, calcium ion. Calcium ion is involved in modulation of many biological phenomena, including contraction of muscles such as skeletal, smooth, and cardiac muscles; activation of movement, phagocytosis, and the like of leukocytes; activation of shape change, secretion, and the like of platelets; activation of lymphocytes; activation of mast cells including secretion of histamine; cell responses mediated by catecholamine a receptor or acetylcholine receptor; exocytosis; release of transmitter substances from neuron terminals; and axoplasmic flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have several calcium ion-binding sites and are believed to be derived from a common origin in terms of molecular evolution. There are also many known calcium-binding motifs. Such well-known motifs include, for example, cadherin domains, EF-hand of calmodulin, C2 domain of Protein kinase C, Gla domain of blood coagulation protein Factor IX, C-type lectins of acyaroglycoprotein receptor and mannose-binding receptor, A domains of LDL receptors, annexin, thrombospondin type 3 domain, and EGF-like domains.

[0633] In the present invention, when the metal ion is calcium ion, the conditions of calcium ion concentration include low calcium ion concentrations and high calcium ion concentrations. “The binding activity varies depending on calcium ion concentrations” means that the antigen-binding activity of an antigen-binding molecule varies due to the difference in the conditions between low and high calcium ion concentrations. For example, the antigen-binding activity of an antigen-binding molecule may be higher at a high calcium ion concentration than at a low calcium ion concentration. Alternatively, the antigen-binding activity of an antigen-binding molecule may be higher at a low calcium ion concentration than at a high calcium ion concentration.

[0634] Herein, the high calcium ion concentration is not particularly limited to a specific value; however, the concentration may preferably be selected between 100 μM and 10 mM. In another embodiment, the concentration may be selected between 200 μM and 5 mM. In an alternative embodiment, the concentration may be selected between 500 μM and 2.5 mM. In still another embodiment, the concentration may be selected between 200 μM and 2 mM. Furthermore, the concentration may be selected between 400 μM and 1.5 mM. In particular, a concentration selected between 500 μM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ion in vivo, is preferred.

[0635] Herein, the low calcium ion concentration is not particularly limited to a specific value; however, the concentration may preferably be selected between 0.1 μM and 30 μM. In another embodiment, the concentration may be selected between 0.2 μM and 20 μM. In still another embodiment, the concentration may be selected between 0.5 μM and 10 μM. In an alternative embodiment, the concentration may be selected between 1 μM and 5 μM. Furthermore, the concentration may be selected between 2 μM and 4 μM. In particular, a concentration selected between 1 μM and 5 μM, which is close to the concentration of ionized calcium in early endosomes in vivo, is preferred.

[0636] Herein, “the antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration” means that the antigen-binding activity of an antigen-binding molecule is weaker at a calcium ion concentration selected between 0.1 μM and 30 μM than at a calcium ion concentration selected between 100 μM and 10 mM. Preferably, it means that the antigen-binding activity of an antigen-binding molecule is weaker at a calcium ion concentration selected between 0.5 μM and 10 μM than at a calcium ion concentration selected between 200 μM and 5 mM. It particularly preferably means that the antigen-binding activity at the calcium ion concentration in the early endosome in vivo is weaker than that at the in vivo plasma calcium ion concentration; and specifically, it means that the antigen-binding activity of an antigen-binding molecule is weaker at a calcium ion concentration selected between 1 μM and 5 μM than at a calcium ion concentration selected between 500 μM and 2.5 mM.

[0637] Whether the antigen-binding activity of an antigen-binding molecule is changed depending on metal ion concentrations can be determined, for example, by the use of known measurement methods such as those described in the section “Binding Activity” above. For example, in order to confirm that the antigen-binding activity of an antigen-binding molecule becomes higher at a high calcium ion concentration than at a low calcium ion concentration, the antigen-binding activity of the antigen-binding molecule at low and high calcium ion concentrations is compared.

[0638] In the present invention, the expression “the antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration” can also be expressed as “the antigen-binding activity of an antigen-binding molecule is higher at a high calcium ion concentration than at a low calcium ion concentration”. In the present invention, “the antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration” is sometimes written as “the antigen-binding ability is weaker at a low calcium ion concentration than at a high calcium ion concentration”. Also, “the antigen-binding activity at a low calcium ion concentration is reduced to be lower than that at a high calcium ion concentration” may be written as “the antigen-binding ability at a low calcium ion concentration is made weaker than that at a high calcium ion concentration”.

[0639] When determining the antigen-binding activity, the conditions other than calcium ion concentration can be appropriately selected by those skilled in the art, and are not particularly limited. For example, the activity can be determined at 37° C. in HEPES buffer. For example, Biacore (GE Healthcare) or such can be used for the determination. When the antigen is a soluble antigen, the antigen-binding activity of an antigen-binding molecule can be assessed by flowing the antigen as an analyte over a chip onto which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of an antigen-binding molecule to the membrane antigen can be assessed by flowing the antigen-binding molecule as an analyte over a chip onto which the antigen is immobilized.

[0640] As long as the antigen-binding activity of an antigen-binding molecule of the present invention is weaker at a low calcium ion concentration than at a high calcium ion concentration, the ratio of the antigen-binding activity between low and high calcium ion concentrations is not particularly limited. However, the ratio of the KD (dissociation constant) of the antigen-binding molecule for an antigen at a low calcium ion concentration with respect to the KD at a high calcium ion concentration, i.e. the value of KD (3 μM Ca) / KD (2 mM Ca), is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (3 μM Ca) / KD (2 mM Ca) value is not particularly limited, and may be any value such as 400, 1000, or 10000 as long as the molecule can be produced by techniques known to those skilled in the art. Furthermore, it may also be specified by the KD (Ca 3 μM) / KD (Ca 1.2 mM) value. Specifically, the KD (Ca 3 μM) / KD (Ca 1.2 mM) value is 2 or greater, preferably the KD (Ca 3 μM) / KD (Ca 1.2 mM) value is 10 or greater, and more preferably the KD (Ca 3 μM) / KD (Ca 1.2 mM) value is 40 or greater. The upper limit of the KD (Ca 3 μM) / KD (Ca 1.2 mM) value is not particularly limited, and may be any value such as 400, 1000, or 10000 as long as the molecule can be produced by techniques known to those skilled in the art.

[0641] When the antigen is a soluble antigen, KD (dissociation constant) can be used to represent the antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, apparent KD (apparent dissociation constant) can be used to represent the activity. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard plot, or flow cytometer.

[0642] Alternatively, for example, the dissociation rate constant (kd) can also be preferably used as an index to represent the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention between low and high calcium concentrations. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index to represent the binding activity ratio, the ratio of the dissociation rate constant (kd) between low and high calcium concentrations, i.e. the value of kd (low calcium concentration) / kd (high calcium concentration), is preferably 2 or more, more preferably 5 or more, still more preferably 10 or more, and yet more preferably 30 or more. The upper limit of the Kd (low calcium concentration) / kd (high calcium concentration) value is not particularly limited, and can be any value such as 50, 100, or 200 as long as the molecule can be produced by techniques known to those skilled in the art.

[0643] When the antigen is a soluble antigen, kd (dissociation rate constant) can be used to represent the antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, apparent kd (apparent dissociation rate constant) can be used to represent the antigen-binding activity. The kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare) or flow cytometer. In the present invention, when the antigen-binding activity of an antigen-binding molecule is determined at different calcium ion concentrations, it is preferable to use the same conditions except for the calcium concentrations.

[0644] For example, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained via screening of antigen-binding domains or antibodies including the steps of:

[0645] (a) determining the antigen-binding activity of an antigen-binding domain or antibody at a low calcium concentration;

[0646] (b) determining the antigen-binding activity of an antigen-binding domain or antibody at a high calcium concentration; and

[0647] (c) selecting an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.

[0648] Moreover, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained via screening of antigen-binding domains or antibodies, or a library thereof, including the steps of:

[0649] (a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof at a high calcium concentration;

[0650] (b) incubating at a low calcium concentration an antigen-binding domain or antibody that has bound to the antigen in step (a); and

[0651] (c) isolating an antigen-binding domain or antibody dissociated in step (b).

[0652] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained via screening of antigen-binding domains or antibodies, or a library thereof, including the steps of:

[0653] (a) contacting an antigen with a library of antigen-binding domains or antibodies at a low calcium concentration;

[0654] (b) selecting an antigen-binding domain or antibody which does not bind to the antigen in step (a);

[0655] (c) allowing the antigen-binding domain or antibody selected in step (c) to bind to the antigen at a high calcium concentration; and

[0656] (d) isolating an antigen-binding domain or antibody that has bound to the antigen in step (c).

[0657] In addition, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the steps of:

[0658] (a) contacting at a high calcium concentration a library of antigen-binding domains or antibodies with a column onto which an antigen is immobilized;

[0659] (b) eluting an antigen-binding domain or antibody that has bound to the column in step (a) from the column at a low calcium concentration; and

[0660] (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0661] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the steps of:

[0662] (a) allowing at a low calcium concentration a library of antigen-binding domains or antibodies to pass through a column onto which an antigen is immobilized;

[0663] (b) collecting an antigen-binding domain or antibody that has been eluted without binding to the column in step (a);

[0664] (c) allowing the antigen-binding domain or antibody collected in step (b) to bind to the antigen at a high calcium concentration; and

[0665] (d) isolating an antigen-binding domain or antibody that has bound to the antigen in step (c).

[0666] Moreover, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the steps of:

[0667] (a) contacting an antigen with a library of antigen-binding domains or antibodies at a high calcium concentration;

[0668] (b) obtaining an antigen-binding domain or antibody that has bound to the antigen in step (a);

[0669] (c) incubating at a low calcium concentration the antigen-binding domain or antibody obtained in step (b); and

[0670] (d) isolating an antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criterion for the selection of step (b).

[0671] The above-described steps may be repeated twice or more times. Thus, the present invention provides antigen-binding domains or antibodies whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which are obtained by screening methods that further comprises the step of repeating twice or more times steps (a) to (c) or (a) to (d) in the above-described screening methods. The number of cycles of steps (a) to (c) or (a) to (d) is not particularly limited, but generally is 10 or less.

[0672] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at a low calcium concentration is not particularly limited as long as it is antigen-binding activity at an ionized calcium concentration of between 0.1 μM and 30 μM, but preferably is antigen-binding activity at an ionized calcium concentration of between 0.5 μM and 10 μM. More preferably, it is antigen-binding activity at the ionized calcium concentration in the early endosome in vivo, specifically, between 1 μM and 5 μM. Meanwhile, the antigen-binding activity of an antigen-binding domain or antibody at a high calcium concentration is not particularly limited, as long as it is antigen-binding activity at an ionized calcium concentration of between 100 μM and 10 mM, but preferably is antigen-binding activity at an ionized calcium concentration of between 200 μM and 5 mM. More preferably, it is antigen-binding activity at the ionized calcium concentration in plasma in vivo, specifically, between 0.5 mM and 2.5 mM.

[0673] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Conditions other than the ionized calcium concentration can be determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as a dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate constant (kd), apparent dissociation constant (apparent kd), and such. These can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard plot, or FACS.

[0674] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration is synonymous with the step of selecting an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.

[0675] As long as the antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration, the difference in the antigen-binding activity between high and low calcium concentrations is not particularly limited; however, the antigen-binding activity at a high calcium concentration is preferably twice or more, more preferably 10 times or more, and still more preferably 40 times or more than that at a low calcium concentration.

[0676] Antigen-binding domains or antibodies of the present invention to be screened by the screening methods described above may be any antigen-binding domains and antibodies. For example, it is possible to screen the above-described antigen-binding domains or antibodies. For example, antigen-binding domains or antibodies having natural sequences or substituted amino acid sequences may be screened.Libraries

[0677] In an embodiment, an antigen-binding domain or antibody of the present invention can be obtained from a library that is mainly composed of a plurality of antigen-binding molecules whose sequences are different from one another and whose antigen-binding domains have at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecules depending on ion concentrations. The ion concentrations preferably include, for example, metal ion concentration and hydrogen ion concentration.

[0678] Herein, a “library” refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding their sequences. The sequences of a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules in a library are not identical, but are different from one another.

[0679] Herein, the phrase “sequences are different from one another” in the expression “a plurality of antigen-binding molecules whose sequences are different from one another” means that the sequences of antigen-binding molecules in a library are different from one another. Specifically, in a library, the number of sequences different from one another reflects the number of independent clones with different sequences, and may also be referred to as “library size”. The library size of a conventional phage display library ranges from 106 to 1012. The library size can be increased up to 1014 by the use of known techniques such as ribosome display. However, the actual number of phage particles used in panning selection of a phage library is in general 10-10000 times greater than the library size. This excess multiplicity is also referred to as “the number of library equivalents”, and means that there are 10 to 10,000 individual clones that have the same amino acid sequence. Thus, in the present invention, the phrase “sequences are different from one another” means that the sequences of independent antigen-binding molecules in a library, excluding library equivalents, are different from one another. More specifically, the above means that there are 106 to 1014 antigen-binding molecules whose sequences are different from one another, preferably 107 to 1012 molecules, more preferably 108 to 1011 molecules, and particularly preferably 108 to 1012 molecules whose sequences are different from one another.

[0680] Herein, the phrase “a plurality of” in the expression “a library mainly composed of a plurality of antigen-binding molecules” generally refers to, in the case of, for example, antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors, or viruses of the present invention, a group of two or more types of the substance. For example, when two or more substances are different from one another in a particular characteristic, this means that there are two or more types of the substance. Such examples may include, for example, mutant amino acids observed at specific amino acid positions in an amino acid sequence. For example, when there are two or more antigen-binding molecules of the present invention whose sequences are substantially the same or preferably the same except for flexible residues or except for particular mutant amino acids at hypervariable positions exposed on the surface, there are a plurality of antigen-binding molecules of the present invention. In another example, when there are two or more polynucleotide molecules whose sequences are substantially the same or preferably the same except for nucleotides encoding flexible residues or nucleotides encoding mutant amino acids of hypervariable positions exposed on the surface, there are a plurality of polynucleotide molecules of the present invention.

[0681] In addition, herein, the phrase “mainly composed of” in the expression “a library mainly composed of a plurality of antigen-binding molecules” reflects the number of antigen-binding molecules whose antigen-binding activity varies depending on ion concentrations, among independent clones with different sequences in a library. Specifically, it is preferable that there are at least 104 antigen-binding molecules having such binding activity in a library. More preferably, antigen-binding domains of the present invention can be obtained from a library containing at least 105 antigen-binding molecules having such binding activity. Still more preferably, antigen-binding domains of the present invention can be obtained from a library containing at least 106 antigen-binding molecules having such binding activity. Particularly preferably, antigen-binding domains of the present invention can be obtained from a library containing at least 10′ antigen-binding molecules having such binding activity. Yet more preferably, antigen-binding domains of the present invention can be obtained from a library containing at least 108 antigen-binding molecules having such binding activity. Alternatively, this may also be preferably expressed as the ratio of the number of antigen-binding molecules whose antigen-binding activity varies depending on ion concentrations with respect to the number of independent clones having different sequences in a library. Specifically, antigen-binding domains of the present invention can be obtained from a library in which antigen-binding molecules having such binding activity account for 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, still more preferably 2% to 20%, and particularly preferably 4% to 10% of independent clones with different sequences in the library. In the case of fusion polypeptides, polynucleotide molecules, or vectors, similar expressions may be possible using the number of molecules or the ratio to the total number of molecules. In the case of viruses, similar expressions may also be possible using the number of virions or the ratio to total number of virions.Amino Acids that Alter the Antigen-Binding Activity of Antigen-Binding Domains Depending on Calcium Ion Concentrations

[0682] Antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when the metal ion is calcium ion, it is possible to use preexisting antibodies, preexisting libraries (phage library, etc.), antibodies or libraries prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals, antibodies or libraries obtained by introducing amino acids capable of chelating calcium (for example, aspartic acid and glutamic acid) or unnatural amino acid mutations into the above-described antibodies or libraries (calcium-chelatable amino acids (such as aspartic acid and glutamic acid), libraries with increased content of unnatural amino acids, libraries prepared by introducing calcium-chelatable amino acids (such as aspartic acid and glutamic acid) or unnatural amino acid mutations at particular positions, or the like.

[0683] Examples of the amino acids that alter the antigen-binding activity of antigen-binding molecules depending on ion concentrations as described above may be any types of amino acids as long as the amino acids form a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in details (for example, Springer et al. (Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof (1995) 2, 305-490); Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562); Chauvaux et al. (Biochem. J. (1990) 265, 261-265); Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol. (1990) 2, 410-419); Schaefer et al. (Genomics (1995) 25, 638-643); Economou et al. (EMBO J. (1990) 9, 349-354); Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). Specifically, any known calcium-binding motifs, including type C lectins such as ASGPR, CD23, MBR, and DC-SIGN, can be included in antigen-binding molecules of the present invention. Preferred examples of such preferred calcium-binding motifs also include, in addition to those described above, for example, the calcium-binding motif in the antigen-binding domain of SEQ ID NO: 4, 9, or 10.

[0684] Furthermore, as amino acids that alter the antigen-binding activity of antigen-binding molecules depending on calcium ion concentrations, for example, amino acids having metal-chelating activity may also be preferably used. Examples of such metal-chelating amino acids include, for example, serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

[0685] Positions in the antigen-binding domains at which the above-described amino acids are contained are not particularly limited to particular positions, and may be any positions within the heavy chain variable region or light chain variable region that forms an antigen-binding domain, as long as they alter the antigen-binding activity of antigen-binding molecules depending on calcium ion concentrations. Specifically, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose heavy chain antigen-binding domains contain amino acids that alter the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations. In another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose heavy chain CDR3 domains contain the above-mentioned amino acids. In still another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose heavy chain CDR3 domains contain the above-mentioned amino acids at positions 95, 96, 100a, and / or 101 as indicated according to the Kabat numbering system.

[0686] Meanwhile, in a non-limiting embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain antigen-binding domains contain amino acids that alter the antigen-binding activity of antigen-binding molecules depending on calcium ion concentrations. In another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR1 domains contain the above-mentioned amino acids. In still another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR1 domains contain the above-mentioned amino acids at positions 30, 31, and / or 32 as indicated according to the Kabat numbering system.

[0687] In another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR2 domains contain the above-mentioned amino acid residues. In yet another embodiment, the present invention provides libraries mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR2 domains contain the above-mentioned amino acid residues at position 50 as indicated according to the Kabat numbering system.

[0688] In still another non-limiting embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR3 domains contain the above-mentioned amino acid residues. In an alternative embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chain CDR3 domains contain the above-mentioned amino acid residues at position 92 as indicated according to the Kabat numbering system.

[0689] Furthermore, in a different embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and in which two or three CDRs selected from the above-described light chain CDR1, CDR2, and CDR3 contain the aforementioned amino acid residues. Moreover, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules whose sequences are different from one another and whose light chains contain the aforementioned amino acid residues at any one or more of positions 30, 31, 32, 50, and / or 92 as indicated according to the Kabat numbering system.

[0690] In a particularly preferred embodiment, the framework sequences of the light chain and / or heavy chain variable region of an antigen-binding molecule preferably contain human germ line framework sequences. Thus, in an embodiment of the present invention, when the framework sequences are completely human sequences, it is expected that when such an antigen-binding molecule of the present invention is administered to humans (for example, to treat diseases), it induces little or no immunogenic response. In the above sense, the phrase “containing a germ line sequence” in the present invention means that a part of the framework sequences of the present invention is identical to a part of any human germ line framework sequences. For example, when the heavy chain FR2 sequence of an antigen-binding molecule of the present invention is a combination of heavy chain FR2 sequences of different human germ line framework sequences, such a molecule is also an antigen-binding molecule of the present invention “containing a germ line sequence”.

[0691] Preferred examples of the frameworks include, for example, fully human framework region sequences currently known, which are included in the website of V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ) or others. Those framework region sequences can be appropriately used as a germ line sequence contained in an antigen-binding molecule of the present invention. The germ line sequences may be categorized according to their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798); Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al. (Nat. Genetics (1994) 7, 162-168)). Appropriate germ line sequences can be selected from Vκ, which is grouped into seven subgroups; Vλ, which is grouped into ten subgroups; and VH, which is grouped into seven subgroups.

[0692] Fully human VH sequences preferably include, but are not limited to, for example, VH sequences of:

[0693] subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, and VH1-69);

[0694] subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70);

[0695] subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74);

[0696] subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61);

[0697] subgroup VH5 (VH5-51);

[0698] subgroup VH6 (VH6-1); and

[0699] subgroup VH7 (VH7-4 and VH7-81).

[0700] These are also described in known documents (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)) and such, and thus persons skilled in the art can appropriately design antigen-binding molecules of the present invention based on the information of these sequences. It is also preferable to use other fully human frameworks or framework sub-regions.

[0701] Fully human VK sequences preferably include, but are not limited to, for example:

[0702] A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;

[0703] A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped into subgroup Vk2;

[0704] A11, A27, L2, L6, L10, L16, L20, and L25, grouped into subgroup Vk3;

[0705] B3, grouped into subgroup Vk4;

[0706] B2 (herein also referred to as Vk5-2), grouped into subgroup Vk5; and

[0707] A10, A14, and A26, grouped into subgroup VK6

[0708] (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022); Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

[0709] Fully human VL sequences preferably include, but are not limited to, for example:

[0710] V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22, grouped into subgroup VL1;

[0711] V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, grouped into subgroup VL1;

[0712] V3-2, V3-3, and V3-4, grouped into subgroup VL3;

[0713] V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into subgroup VL4; and

[0714] V5-1, V5-2, V5-4, and V5-6, grouped into subgroup VL5 (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).

[0715] Normally, these framework sequences are different from one another at one or more amino acid residues. These framework sequences can be used in combination with “at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations” of the present invention. Other examples of the fully human frameworks used in combination with “at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations” of the present invention include, but are not limited to, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (for example, Kabat et al. (1991) supra; Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

[0716] Without being bound by a particular theory, one reason for the expectation that the use of germ line sequences precludes adverse immune responses in most individuals is believed to be as follows. As a result of the process of affinity maturation during normal immune responses, somatic mutation occurs frequently in the variable regions of immunoglobulin. Such mutations mostly occur around CDRs whose sequences are hypervariable, but also affect residues of framework regions. Such framework mutations do not exist on the germ line genes, and also they are less likely to be immunogenic in patients. On the other hand, the normal human population is exposed to most of the framework sequences expressed from the germ line genes. As a result of immunotolerance, these germ line frameworks are expected to have low or no immunogenicity in patients. To maximize the possibility of immunotolerance, variable region-encoding genes may be selected from a group of commonly occurring functional germ line genes.

[0717] Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed to produce antigen-binding molecules of the present invention in which the above-described framework sequences contain amino acids that alter the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations.

[0718] For example, a library which contains a plurality of antigen-binding molecules of the present invention whose sequences are different from one another can be constructed by combining heavy chain variable regions prepared as a randomized variable region sequence library with a light chain variable region selected as a framework sequence originally containing at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentrations. Non-limiting examples of such libraries when the ion concentration is calcium ion concentration include a library in which a light chain variable region sequence belonging to the Vk5-2 family represented by the light chain variable region sequence of SEQ ID NO: 4 (Vk5-2) is combined with heavy chain variable regions produced as a randomized variable region sequence library.

[0719] Alternatively, a light chain variable region sequence selected as a framework region originally containing at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule as mentioned above can be design to contain various amino acid residues other than the above amino acid residues. Herein, such residues are referred to as flexible residues. The number and position of flexible residues are not particularly limited as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on ion concentrations. Specifically, the CDR sequences and / or FR sequences of the heavy chain and / or light chain may contain one or more flexible residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence of SEQ ID NO: 4 (Vk5-2) include the amino acid residues listed in Tables 1 or 2.

[0720] TABLE 1KabatCDRnumberingAmino acid in 70% of the totalCDR128S: 100%29I: 100%30E: 72%N: 14%S: 14%31D: 100%32D: 100%33L: 100%34A: 70%N: 30%CDR250E: 100%51A: 100%52S: 100%53H: 5%N: 25%S: 45%T: 25%54L: 100%55Q: 100%56S: 100%CDR390Q: 100%91H: 25%S: 15%R: 15%Y: 45%92D: 80%N: 10%S: 10%93D: 5%G: 10%N: 25%S: 50%R: 10%94S: 50%Y: 50%95P: 100%96L: 50%Y: 50%

[0721] TABLE 2KabatCDRnumberingAmino acid in 30% of the totalCDR128S: 100%29I: 100%30E: 83%S: 17%31D: 100%32D: 100%33L: 100%34A: 70%N: 30%CDR250H: 100%51A: 100%52S: 100%53H: 5%N: 25%S: 45%T: 25%54L: 100%55Q: 100%56S: 100%CDR390Q: 100%91H: 25%S: 15%R: 15%Y: 45%92D: 80%N: 10%S: 10%93D: 5%G: 10%N: 25%S: 50%R: 10%94S: 50%Y: 50%95P: 100%96L: 50%Y: 50%

[0722] Herein, flexible residues refer to amino acid residue variations present at hypervariable positions at which several different amino acids are present on the light chain and heavy chain variable regions when the amino acid sequences of known and / or native antibodies or antigen-binding domains are compared. Hypervariable positions are generally located in the CDR regions. In an embodiment, the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful to determine hypervariable positions in known and / or native antibodies. Furthermore, databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinforg.uk / abs / index.html) provide the collected sequences of many human light chains and heavy chains and their locations. The information on the sequences and locations is useful to determine hypervariable positions in the present invention. According to the present invention, when a certain amino acid position has preferably about 2 to about 20 possible amino acid residue variations, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, and preferably 10 to 12 possible amino acid residue variations, the position is hypervariable. In some embodiments, a certain amino acid position may have preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, and preferably about 12 amino acid residue variations.

[0723] Alternatively, a library containing a plurality of antigen-binding molecules of the present invention whose sequences are different from one another can be constructed by combining heavy chain variable regions produced as a randomized variable region sequence library with light chain variable regions into which at least one amino acid residue that alters the antigen-binding activity of antigen-binding molecules depending on ion concentrations as mentioned above is introduced. When the ion concentration is calcium ion concentration, non-limiting examples of such libraries preferably include, for example, libraries in which heavy chain variable regions produced as a randomized variable region sequence library are combined with light chain variable region sequences in which a particular residue(s) in a germ line sequence such as SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), or SEQ ID NO: 8 (Vk4) has been substituted with at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on calcium ion concentrations. Non-limiting examples of such amino acid residues include amino acid residues in light chain CDR1. Furthermore, non-limiting examples of such amino acid residues include amino acid residues in light chain CDR2. In addition, non-limiting examples of such amino acid residues also include amino acid residues in light chain CDR3.

[0724] Non-limiting examples of such amino acid residues contained in light chain CDR1 include those at positions 30, 31, and / or 32 in the CDR1 of light chain variable region as indicated by Kabat numbering. Furthermore, non-limiting examples of such amino acid residues contained in light chain CDR2 include an amino acid residue at position 50 in the CDR2 of light chain variable region as indicated by Kabat numbering. Moreover, non-limiting examples of such amino acid residues contained in light chain CDR3 include an amino acid residue at position 92 in the CDR3 of light chain variable region as indicated by Kabat numbering. These amino acid residues can be contained alone or in combination as long as they form a calcium-binding motif and / or as long as the antigen-binding activity of an antigen-binding molecule varies depending on calcium ion concentrations. Meanwhile, as troponin C, calmodulin, parvalbumin, and myosin light chain, which have several calcium ion-binding sites and are believed to be derived from a common origin in terms of molecular evolution, are known, the light chain CDR1, CDR2, and / or CDR3 can be designed to have their binding motifs. For example, it is possible to use cadherin domains, EF hand of calmodulin, C2 domain of Protein kinase C, Gla domain of blood coagulation protein FactorIX, C type lectins of acyaroglycoprotein receptor and mannose-binding receptor, A domains of LDL receptors, annexin, thrombospondin type 3 domain, and EGF-like domains in an appropriate manner for the above purposes.

[0725] When heavy chain variable regions produced as a randomized variable region sequence library and light chain variable regions into which at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations has been introduced are combined as described above, the sequences of the light chain variable regions can be designed to contain flexible residues in the same manner as described above. The number and position of such flexible residues are not particularly limited to particular embodiments as long as the antigen-binding activity of antigen-binding molecules of the present invention varies depending on ion concentrations. Specifically, the CDR sequences and / or FR sequences of heavy chain and / or light chain can contain one or more flexible residues. When the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the sequence of light chain variable region include the amino acid residues listed in Tables 1 and 2.

[0726] The preferred heavy chain variable regions to be combined include, for example, randomized variable region libraries. Known methods are combined as appropriate to produce a randomized variable region library. In a non-limiting embodiment of the present invention, an immune library constructed based on antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infections, persons with an elevated antibody titer in blood as a result of vaccination, cancer patients, or auto immune disease patients, may be preferably used as a randomized variable region library.

[0727] In another non-limiting embodiment of the present invention, a synthetic library produced by replacing the CDR sequences of V genes in genomic DNA or functional reshaped V genes with a set of synthetic oligonucleotides containing sequences encoding codon sets of an appropriate length can also be preferably used as a randomized variable region library. In this case, since sequence diversity is observed in the heavy chain CDR3 sequence, it is also possible to replace the CDR3 sequence only. A criterion of giving rise to diversity in amino acids in the variable region of an antigen-binding molecule is that diversity is given to amino acid residues at surface-exposed positions in the antigen-binding molecule. The surface-exposed position refers to a position that is considered to be able to be exposed on the surface and / or contacted with an antigen, based on structure, ensemble of structures, and / or modeled structure of an antigen-binding molecule. In general, such positions are CDRs. Preferably, surface-exposed positions are determined using coordinates from a three-dimensional model of an antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (for example, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from an antibody. Software that can be used for these purposes preferably includes SYBYL Biopolymer Module software (Tripos Associates). Generally or preferably, when an algorithm requires a user input size parameter, the “size” of a probe which is used in the calculation is set at about 1.4 Angstrom or smaller in radius. Furthermore, methods for determining surface-exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46-53).

[0728] In another non-limiting embodiment of the present invention, a naive library, which is constructed from antibody genes derived from lymphocytes of healthy persons and whose repertoire consists of naive sequences, which are antibody sequences with no bias, can also be particularly preferably used as a randomized variable region library (Gejima et al. (Human Antibodies (2002) 11, 121-129); Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)). Herein, an amino acid sequence comprising a naive sequence refers to an amino acid sequence obtained from such a naive library.

[0729] In one embodiment of the present invention, an antigen-binding domain of the present invention can be obtained from a library containing a plurality of antigen-binding molecules of the present invention whose sequences are different from one another, prepared by combining light chain variable regions constructed as a randomized variable region sequence library with a heavy chain variable region selected as a framework sequence that originally contains “at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations”. When the ion concentration is calcium ion concentration, non-limiting examples of such libraries preferably include those constructed by combining light chain variable regions constructed as a randomized variable region sequence library with the sequence of heavy chain variable region of SEQ ID NO: 9 (6RL #9-IgG1) or SEQ ID NO: 10 (6KC4-1 #85-IgG1). Alternatively, such a library can be constructed by selecting appropriate light chain variable regions from those having germ line sequences, instead of light chain variable regions constructed as a randomized variable region sequence library. Such preferred libraries include, for example, those in which the sequence of heavy chain variable region of SEQ ID NO: 9 (6RL #9-IgG1) or SEQ ID NO: 10 (6KC4-1 #85-IgG1) is combined with light chain variable regions having germ line sequences.

[0730] Alternatively, the sequence of a heavy chain variable region selected as a framework sequence that originally contains “at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule” as mentioned above can be designed to contain flexible residues. The number and position of the flexible residues are not particularly limited as long as the antigen-binding activity of an antigen-binding molecule of the present invention varies depending on ion concentrations. Specifically, the CDR and / or FR sequences of heavy chain and / or light chain can contain one or more flexible residues. When the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the sequence of heavy chain variable region of SEQ ID NO: 9 (6RL #9-IgG1) include all amino acid residues of heavy chain CDR1 and CDR2 and the amino acid residues of the heavy chain CDR3 except those at positions 95, 96, and / or 100a as indicated by Kabat numbering. Alternatively, non-limiting examples of flexible residues to be introduced into the sequence of heavy chain variable region of SEQ ID NO: 10 (6KC4-1 #85-IgG1) include all amino acid residues of heavy chain CDR1 and CDR2 and the amino acid residues of the heavy chain CDR3 except those at amino acid positions 95 and / or 101 as indicated by Kabat numbering.

[0731] Alternatively, a library containing a plurality of antigen-binding molecules whose sequences are different from one another can be constructed by combining light chain variable regions constructed as a randomized variable region sequence library or light chain variable regions having germ line sequences with heavy chain variable regions into which “at least one amino acid residue responsible for the ion concentration-dependent change in the antigen-binding activity of an antigen-binding molecule” has been introduced as mentioned above. When the ion concentration is calcium ion concentration, non-limiting examples of such libraries preferably include those in which light chain variable regions constructed as a randomized variable region sequence library or light chain variable regions having germ line sequences are combined with the sequence of a heavy chain variable region in which a particular residue(s) has been substituted with at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on calcium ion concentrations. Non-limiting examples of such amino acid residues include amino acid residues of the heavy chain CDR1. Further non-limiting examples of such amino acid residues include amino acid residues of the heavy chain CDR2. In addition, non-limiting examples of such amino acid residues also include amino acid residues of the heavy chain CDR3. Non-limiting examples of such amino acid residues of heavy chain CDR3 include the amino acids of positions 95, 96, 100a, and / or 101 in the CDR3 of heavy chain variable region as indicated by the Kabat numbering. Furthermore, these amino acid residues can be contained alone or in combination as long as they form a calcium-binding motif and / or the antigen-binding activity of an antigen-binding molecule varies depending on calcium ion concentrations.

[0732] When light chain variable regions constructed as a randomized variable region sequence library or light chain variable regions having germ line sequence are combined with a heavy chain variable region into which at least one amino acid residue that alter the antigen-binding activity of an antigen-binding molecule depending on ion concentrations as mentioned above has been introduced, the sequence of the heavy chain variable region can also be designed to contain flexible residues in the same manner as described above. The number and position of flexible residues are not particularly limited as long as the antigen-binding activity of an antigen-binding molecule of the present invention varies depending on ion concentrations. Specifically, the heavy chain CDR and / or FR sequences may contain one or more flexible residues.

[0733] Furthermore, randomized variable region libraries can be preferably used as amino acid sequences of CDR1, CDR2, and / or CDR3 of the heavy chain variable region other than the amino acid residues that alter the antigen-binding activity of an antigen-binding molecule. When germ line sequences are used as light chain variable regions, non-limiting examples of such sequences include those of SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), and SEQ ID NO: 8 (Vk4).

[0734] Any of the above-described amino acids that alter the antigen-binding activity of an antigen-binding molecule depending on calcium ion concentrations can be preferably used, as long as they form a calcium-binding motif. Specifically, such amino acids include electron-donating amino acids. Preferred examples of such electron-donating amino acids include, serine, threonine, asparagine, glutamic acid, aspartic acid, and glutamic acid.Condition of Hydrogen Ion Concentrations

[0735] In an embodiment of the present invention, the condition of ion concentrations refers to the condition of hydrogen ion concentrations or pH condition. In the present invention, the concentration of proton, i.e., the nucleus of hydrogen atom, is treated as synonymous with hydrogen index (pH). When the activity of hydrogen ion in an aqueous solution is represented as aH+, pH is defined as −log 10aH+. When the ionic strength of the aqueous solution is low (for example, lower than 10−3), aH+ is nearly equal to the hydrogen ion strength. For example, the ionic product of water at 25° C. and 1 atmosphere is Kw=aH+aOH=10−14, and therefore in pure water, aH+=aOH=10−7. In this case, pH=7 is neutral; an aqueous solution whose pH is lower than 7 is acidic or whose pH is greater than 7 is alkaline.

[0736] In the present invention, when pH condition is used as the ion concentration condition, pH conditions include high hydrogen ion concentrations or low pHs, i.e., an acidic pH range, and low hydrogen ion concentrations or high pHs, i.e., a neutral pH range. “The binding activity varies depending on pH condition” means that the antigen-binding activity of an antigen-binding molecule varies due to the difference in conditions of a high hydrogen ion concentration or low pH (an acidic pH range) and a low hydrogen ion concentration or high pH (a neutral pH range). This includes, for example, the case where the antigen-binding activity of an antigen-binding molecule is higher in a neutral pH range than in an acidic pH range and the case where the antigen-binding activity of an antigen-binding molecule is higher in an acidic pH range than in a neutral pH range.

[0737] Herein, neutral pH range is not limited to a specific value and is preferably selected from between pH 6.7 and pH 10.0. In another embodiment, the pH can be selected from between pH 6.7 and pH 9.5. In still another embodiment, the pH can be selected from between pH 7.0 and pH 9.0. In yet another embodiment, the pH can be selected from between pH 7.0 and pH 8.0. In particular, the preferred pH includes pH 7.4, which is close to the pH of plasma (blood) in vivo.

[0738] Herein, an acidic pH range is not limited to a specific value and is preferably selected from between pH 4.0 and pH 6.5. In another embodiment, the pH can be selected from between pH 4.5 and pH 6.5. In still another embodiment, the pH can be selected from between pH 5.0 and pH 6.5. In yet another embodiment, the pH can be selected from between pH 5.5 and pH 6.5. In particular, the preferred pH includes pH 5.8, which is close to the ionized calcium concentration in the early endosome in vivo.

[0739] In the present invention, “the antigen-binding activity of an antigen-binding molecule at a high hydrogen ion concentration or low pH (an acidic pH range) is lower than that at a low hydrogen ion concentration or high pH (a neutral pH range)” means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 4.0 and pH 6.5 is weaker than that at a pH selected from between pH 6.7 and pH 10.0; preferably means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH4.5 and pH 6.5 is weaker than that at a pH selected from between pH 6.7 and pH 9.5; more preferably, means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 5.0 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 9.0; still more preferably means that the antigen-binding activity of an antigen-binding molecule at a pH selected from between pH 5.5 and pH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH 8.0; particularly preferably means that the antigen-binding activity at the pH in the early endosome in vivo is weaker than the antigen-binding activity at the pH of plasma in vivo; and specifically means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.

[0740] Whether the antigen-binding activity of an antigen-binding molecule has changed by the pH condition can be determined, for example, by the use of known measurement methods such as those described in the section “Binding Activity” above. Specifically, the binding activity is measured under different pH conditions using the measurement methods described above. For example, the antigen-binding activity of an antigen-binding molecule is compared under the conditions of acidic pH range and neutral pH range to confirm that the antigen-binding activity of the antigen-binding molecule changes to be higher under the condition of neutral pH range than that under the condition of acidic pH range.

[0741] Furthermore, in the present invention, the expression “the antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range” can also be expressed as “the antigen-binding activity of an antigen-binding molecule at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is higher than that at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range”. In the present invention, “the antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range” may be described as “the antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is weaker than the antigen-binding ability at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range”. Alternatively, “the antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is reduced to be lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range” may be described as “the antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is reduced to be weaker than the antigen-binding ability at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range”.

[0742] The conditions other than hydrogen ion concentration or pH for measuring the antigen-binding activity may be suitably selected by those skilled in the art and are not particularly limited. Measurements can be carried out, for example, at 37° C. using HEPES buffer. Measurements can be carried out, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the antigen-binding activity of an antigen-binding molecule can be determined by assessing the binding activity to the soluble antigen by pouring the antigen as an analyte into a chip immobilized with the antigen-binding molecule. When the antigen is a membrane antigen, the binding activity to the membrane antigen can be assessed by pouring the antigen-binding molecule as an analyte into a chip immobilized with the antigen.

[0743] As long as the antigen-binding activity of an antigen-binding molecule of the present invention at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range is weaker than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, the ratio of the antigen-binding activity between that at a high hydrogen ion concentration or low pH, i.e., an acidic pH range, and at a low hydrogen ion concentration or high pH, i.e., a neutral pH range is not particularly limited, and the value of KD (pH 5.8) / KD (pH 7.4), which is the ratio of the dissociation constant (KD) for an antigen at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range to the KD at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is preferably 2 or more; more preferably the value of KD (pH 5.8) / KD (pH 7.4) is 10 or more; and still more preferably the value of KD (pH 5.8) / KD (pH 7.4) is 40 or more. The upper limit of KD (pH 5.8) / KD (pH 7.4) value is not particularly limited, and may be any value such as 400, 1000, or 10000, as long as the molecule can be produced by the techniques of those skilled in the art.

[0744] When the antigen is a soluble antigen, the dissociation constant (KD) can be used as the value for antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. The dissociation constant (KD) and apparent dissociation constant (KD) can be measured by methods known to those skilled in the art, and Biacore (GE healthcare), Scatchard plot, flow cytometer, and such can be used.

[0745] Alternatively, for example, the dissociation rate constant (kd) can be suitably used as an index for indicating the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention between that at a high hydrogen ion concentration or low pH, i.e., an acidic pH range and a low hydrogen ion concentration or high pH, i.e., a neutral pH range. When kd (dissociation rate constant) is used as an index for indicating the binding activity ratio instead of KD (dissociation constant), the value of kd (in an acidic pH range) / kd (in a neutral pH range), which is the ratio of kd (dissociation rate constant) for the antigen at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range to kd (dissociation rate constant) at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is preferably 2 or more, more preferably 5 or more, still more preferably 10 or more, and yet more preferably 30 or more. The upper limit of kd (in an acidic pH range) / kd (in a neutral pH range) value is not particularly limited, and may be any value such as 50, 100, or 200, as long as the molecule can be produced by the techniques of those skilled in the art.

[0746] When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the value for antigen-binding activity and when the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. The dissociation rate constant (kd) and apparent dissociation rate constant (kd) can be determined by methods known to those skilled in the art, and Biacore (GE healthcare), flow cytometer, and such may be used. In the present invention, when the antigen-binding activity of an antigen-binding molecule is measured at different hydrogen ion concentrations, i.e., pHs, conditions other than the hydrogen ion concentration, i.e., pH, are preferably the same.

[0747] For example, an antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained via screening of antigen-binding domains or antibodies, comprising the following steps (a) to (c):

[0748] (a) obtaining the antigen-binding activity of an antigen-binding domain or antibody in an acidic pH range;

[0749] (b) obtaining the antigen-binding activity of an antigen-binding domain or antibody in a neutral pH range; and

[0750] (c) selecting an antigen-binding domain or antibody whose antigen-binding activity in the acidic pH range is lower than that in the neutral pH range.

[0751] Alternatively, an antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is one embodiment provided by the present invention, can be obtained via screening of antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (c):

[0752] (a) contacting an antigen-binding domain or antibody, or a library thereof, in a neutral pH range with an antigen;

[0753] (b) placing in an acidic pH range the antigen-binding domain or antibody bound to the antigen in step (a); and

[0754] (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0755] An antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is another embodiment provided by the present invention, can be obtained via screening of antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d):

[0756] (a) contacting in an acidic pH range an antigen with a library of antigen-binding domains or antibodies;

[0757] (b) selecting the antigen-binding domain or antibody which does not bind to the antigen in step (a);

[0758] (c) allowing the antigen-binding domain or antibody selected in step (b) to bind with the antigen in a neutral pH range; and

[0759] (d) isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0760] An antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is even another embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (c):

[0761] (a) contacting in a neutral pH range a library of antigen-binding domains or antibodies with a column immobilized with an antigen;

[0762] (b) eluting in an acidic pH range from the column the antigen-binding domain or antibody bound to the column in step (a); and

[0763] (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0764] An antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH, range is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is still another embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (d):

[0765] (a) allowing, in an acidic pH range, a library of antigen-binding domains or antibodies to pass a column immobilized with an antigen;

[0766] (b) collecting the antigen-binding domain or antibody eluted without binding to the column in step (a);

[0767] (c) allowing the antigen-binding domain or antibody collected in step (b) to bind with the antigen in a neutral pH range; and

[0768] (d) isolating the antigen-binding domain or antibody bound to the antigen in step (c).

[0769] An antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, which is yet another embodiment provided by the present invention, can be obtained by a screening method comprising the following steps (a) to (d):

[0770] (a) contacting an antigen with a library of antigen-binding domains or antibodies in a neutral pH range;

[0771] (b) obtaining the antigen-binding domain or antibody bound to the antigen in step (a);

[0772] (c) placing in an acidic pH range the antigen-binding domain or antibody obtained in step (b); and

[0773] (d) isolating the antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criterion for the selection in step (b).

[0774] The above-described steps may be repeated twice or more times. Thus, the present invention provides antigen-binding domains and antibodies whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range, which are obtained by a screening method that further comprises the steps of repeating steps (a) to (c) or (a) to (d) in the above-described screening methods. The number of times that steps (a) to (c) or (a) to (d) is repeated is not particularly limited; however, the number is 10 or less in general.

[0775] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is not particularly limited, as long as it is the antigen-binding activity at a pH of between 4.0 and 6.5, and includes the antigen-binding activity at a pH of between 4.5 and 6.6 as the preferred pH. The antigen-binding activity also includes that at a pH of between 5.0 and 6.5, and that at a pH of between 5.5 and 6.5 as another preferred pH. The antigen-binding activity also includes that at the pH in the early endosome in vivo as the more preferred pH, and specifically, that at pH 5.8. Meanwhile, the antigen-binding activity of an antigen-binding domain or antibody at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is not particularly limited, as long as it is the antigen-binding activity at a pH of between 6.7 and 10, and includes the antigen-binding activity at a pH of between 6.7 and 9.5 as the preferred pH. The antigen-binding activity also includes that at a pH of between 7.0 and 9.5 and that at a pH of between 7.0 and 8.0 as another preferred pH. The antigen-binding activity also includes that at the pH of plasma in vivo as the more preferred pH, and specifically, that at pH 7.4.

[0776] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Those skilled in the art can suitably determine conditions other than ionized calcium concentration. The antigen-binding activity of an antigen-binding domain or antibody can be assessed based on the dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), apparent dissociation rate constant (kd), and such. These can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), Scatchard plot, or FACS.

[0777] Herein, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is higher than that at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is synonymous with the step of selecting an antigen-binding domain or antibody whose antigen-binding activity at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, is lower than that at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range.

[0778] As long as the antigen-binding activity at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is higher than that at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, the difference between the antigen-binding activity at a low hydrogen ion concentration or high pH, i.e., a neutral pH range, and that at a high hydrogen ion concentration or low pH, i.e., an acidic pH range, is not particularly limited; however, the antigen-binding activity at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is preferably twice or more, more preferably 10 times or more, and still more preferably 40 times or more than that at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range.

[0779] The antigen binding domain or antibody of the present invention screened by the screening methods described above may be any antigen-binding domain or antibody, and the above-mentioned antigen-binding domain or antibody may be screened. For example, antigen-binding domain or antibody having the native sequence may be screened, and antigen-binding domain or antibody in which their amino acid sequences have been substituted may be screened.

[0780] The antigen-binding domain or antibody of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries prepared from B cells of immunized animals or from hybridomas obtained by immunizing animals, antibodies or libraries (libraries with increased content of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids, libraries introduced with amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acid mutations at specific positions, etc.) obtained by introducing amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acid mutations into the above-described antibodies or libraries may be used.

[0781] Methods for obtaining an antigen-binding domain or antibody whose antigen-binding activity at a low hydrogen ion concentration or high pH, i.e., in a neutral pH range, is higher than that at a high hydrogen ion concentration or low pH, i.e., in an acidic pH range, from an antigen-binding domains or antibodies prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals preferably include, for example, the antigen-binding molecule or antibody in which at least one of the amino acids of the antigen-binding domain or antibody is substituted with an amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or an unnatural amino acid mutation, or the antigen-binding domain or antibody inserted with an amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acid, such as those described in WO 2009 / 125825.

[0782] The sites of introducing mutations of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids are not particularly limited, and may be any position as long as the antigen-binding activity in an acidic pH range becomes weaker than that in a neutral pH range (the value of KD (in an acidic pH range) / KD (in a neutral pH range) or kd (in an acidic pH range) / kd (in a neutral pH range) is increased) as compared to before substitution or insertion. For example, when the antigen-binding molecule is an antibody, antibody variable region and CDRs are suitable. Those skilled in the art can appropriately determine the number of amino acids to be substituted with or the number of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids to be inserted. It is possible to substitute with a single amino acid having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or a single unnatural amino acid; it is possible to insert a single amino acid having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or a single unnatural amino acid; it is possible to substitute with two or more amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or two or more unnatural amino acids; and it is possible to insert two or more amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or two or more unnatural amino acids. Alternatively, other amino acids can be deleted, added, inserted, and / or substituted concomitantly, aside from the substitution into amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids, or the insertion of amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids. Substitution into or insertion of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids can performed randomly by methods such as histidine scanning, in which the alanine of alanine scanning known to those skilled in the art is replaced with histidine. Antigen-binding molecules exhibiting a greater value of KD (in an acidic pH range) / KD (in a neutral pH range) or kd (in an acidic pH range) / kd (in a neutral pH range) as compared to before the mutation can be selected from antigen-binding domains or antibodies introduced with random insertions or substitution mutations of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids.

[0783] Preferred examples of antigen-binding molecules containing the mutation into amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids as described above and whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range include, antigen-binding molecules whose antigen-binding activity in the neutral pH range after the mutation into amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids is comparable to that before the mutation into amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids. Herein, “an antigen-binding molecule after the mutation with amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids has an antigen-binding activity comparable to that before the mutation with amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids” means that, when taking the antigen-binding activity of an antigen-binding molecule before the mutation with amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids as 100%, the antigen-binding activity of an antigen-binding molecule after the mutation with amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids is at least 10% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more. The antigen-binding activity after the mutation of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids at pH 7.4 may be higher than that before the mutation of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids at pH 7.4. If the antigen-binding activity of an antigen-binding molecule is decreased due to insertion of or substitution into amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids, the antigen-binding activity can be made to be comparable to that before the insertion of or substitution into amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids, by introducing a substitution, deletion, addition, and / or insertion of one or more amino acids of the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity has been adjusted to be comparable by substitution, deletion, addition, and / or insertion of one or more amino acids after substitution or insertion of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids. Meanwhile, when an antigen-binding molecule is a substance containing an antibody constant region, preferred embodiments of antigen-binding molecules whose antigen-binding activity at an acidic pH range is lower than that in a neutral pH range include methods in which the antibody constant regions contained in the antigen-binding molecules have been modified. Specific examples of modified antibody constant regions preferably include the constant regions of SEQ ID NOs: 11, 12, 13, and 14.Amino Acids that Alter the Antigen-Binding Activity of Antigen-Binding Domain Depending on the Hydrogen Ion Concentration Conditions

[0784] Antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when ion concentration condition is hydrogen ion concentration condition or pH condition, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries prepared from B cells of immunized animals or from hybridomas obtained by immunizing animals, antibodies or libraries (libraries with increased content of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids, libraries introduced with mutations of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids at specific positions, etc.) obtained by introducing mutations of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acids into the above-described antibodies or libraries may be used.

[0785] In one non-limiting embodiment of the present invention, a library containing multiple antigen-binding molecules of the present invention whose sequences are different from one another can also be constructed by combining heavy chain variable regions, produced as a randomized variable region sequence library, with light chain variable regions introduced with “at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on the hydrogen ion concentration condition”.

[0786] Such amino acid residues include, but are not limited to, for example, amino acid residues contained in the light chain CDR1. The amino acid residues also include, but are not limited to, for example, amino acid residues contained in the light chain CDR2. The amino acid residues also include, but are not limited to, for example, amino acid residues contained in the light chain CDR3.

[0787] The above-described amino acid residues contained in the light chain CDR1 include, but are not limited to, for example, amino acid residues of positions 24, 27, 28, 31, 32, and / or 34 according to Kabat numbering in the CDR1 of light chain variable region. Meanwhile, the amino acid residues contained in the light chain CDR2 include, but are not limited to, for example, amino acid residues of positions 50, 51, 52, 53, 54, 55, and / or 56 according to Kabat numbering in the CDR2 of light chain variable region. Furthermore, the amino acid residues in the light chain CDR3 include, but are not limited to, for example, amino acid residues of positions 89, 90, 91, 92, 93, 94, and / or 95A according to Kabat numbering in the CDR3 of light chain variable region. Moreover, the amino acid residues can be contained alone or can be contained in combination of two or more amino acids as long as they allow the change in the antigen-binding activity of an antigen-binding molecule depending on the hydrogen ion concentration.

[0788] Even when the heavy chain variable region produced as a randomized variable region sequence library is combined with the above-described light chain variable region introduced with “at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on the hydrogen ion concentration condition”, it is possible to design so that the flexible residues are contained in the sequence of the light chain variable region in the same manner as described above. The number and position of the flexible residues are not particularly limited to a specific embodiment, as long as the antigen-binding activity of an antigen-binding molecule of the present invention changes depending on the hydrogen ion concentration condition. Specifically, the CDR and / or FR sequences of heavy chain and / or light chain can contain one or more flexible residues. For example, flexible residues to be introduced into the sequences of the light chain variable regions include, but are not limited to, for example, the amino acid residues listed in Tables 3 and 4. Meanwhile, amino acid sequences of light chain variable regions other than the flexible residues and amino acid residues that change the antigen-binding activity of an antigen-binding molecule depending on the hydrogen ion concentration condition suitably include, but are not limited to, germ line sequences such as Vk1 (SEQ ID NO: 5), Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7), and Vk4 (SEQ ID NO: 8).

[0789] TABLE 3POSITIONAMINO ACIDCDR128S: 100%29I: 100%30N: 25%S: 25%R: 25%H: 25%31S: 100%32H: 100%33L: 100%34A: 50%N: 50%CDR250H: 100%ORA: 25%D: 25%G: 25%K: 25%51A: 100%A: 100%52S: 100%S: 100%53K: 33.3%N: 33.3%S: 33.3%H: 100%54L: 100%L: 100%55Q: 100%Q: 100%56S: 100%S: 100%CDR390Q: 100%ORQ: 100%91H: 100%S: 33.3%R: 33.3%Y: 33.3%92G: 25%N: 25%S: 25%Y: 25%H: 100%93H: 33.3%N: 33.3%S: 33.3%H: 33.3%N: 33.3%S: 33.3%94S: 50%Y: 50%S: 50%Y: 50%95P: 100%P: 100%96L: 50%Y: 50%L: 50%Y: 50%(Position indicates Kabat numbering)

[0790] TABLE 4CDRPOSITIONAMINO ACIDCDR128S: 100%29I: 100%30H: 30%N: 10%S: 50%R: 10%31N: 35%S: 65%32H: 40%N: 20%Y: 40%33L: 100%34A: 70%N: 30%CDR250A: 25%D: 15%G: 25%H: 30%K: 5%51A: 100%52S: 100%53H: 30%K: 10%N: 15%S: 45%54L: 100%55Q: 100%56S: 100%CDR390Q: 100%91H: 30%S: 15%R: 10%Y: 45%92G: 20%H: 30%N: 20%S: 15%Y: 15%93H: 30%N: 25%S: 45%94S: 50%Y: 50%95P: 100%96L: 50%Y: 50%(Position indicates Kabat numbering)

[0791] Any amino acid residue may be suitably used as the above-described amino acid residues that change the antigen-binding activity of an antigen-binding molecule depending on the hydrogen ion concentration condition. Specifically, such amino acid residues include amino acids with a side chain pKa of 4.0-8.0. Such electron-releasing amino acids preferably include, for example, naturally occurring amino acids such as histidine and glutamic acid, as well as unnatural amino acids such as histidine analogs (US20090035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768). Particularly preferred amino acid residues include, for example, amino acids with a side chain pKa of 6.0-7.0. Such electron-releasing amino acid residues preferably include, for example, histidine.

[0792] Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR can be appropriately employed to modify the amino acids of antigen-binding domains. Furthermore, various known methods can also be used as an amino acid modification method for substituting amino acids by those other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which amber suppressor tRNA, which is complementary to UAG codon (amber codon) that is a stop codon, is linked with an unnatural amino acid may be suitably used.

[0793] The preferred heavy chain variable region that is used in combination includes, for example, randomized variable region libraries. Known methods are appropriately combined as a method for producing a randomized variable region library. In a non-limiting embodiment of the present invention, an immune library constructed based on antibody genes derived from animals immunized with specific antigens, patients with infection or persons with an elevated antibody titer in blood as a result of vaccination, cancer patients, or lymphocytes of auto immune diseases may be suitably used as a randomized variable region library.

[0794] In another non-limiting embodiment of the present invention, in the same manner as described above, a synthetic library in which the CDR sequences of V genes from genomic DNA or functional reconstructed V genes are replaced with a set of synthetic oligonucleotides containing the sequences encoding codon sets of an appropriate length can also be suitably used as a randomized variable region library. In this case, the CDR3 sequence alone may be replaced because variety in the gene sequence of heavy chain CDR3 is observed. The basis for giving rise to amino acid variations in the variable region of an antigen-binding molecule is to generate variations of amino acid residues of surface-exposed positions of the antigen-binding molecule. The surface-exposed position refers to a position where an amino acid is exposed on the surface and / or contacted with an antigen based on the conformation, structural ensemble, and / or modeled structure of an antigen-binding molecule, and in general, such positions are the CDRs. The surface-exposed positions are preferably determined using the coordinates derived from a three-dimensional model of the antigen-binding molecule using computer programs such as InsightII program (Accelrys). The surface-exposed positions can be determined using algorithms known in the art (for example, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). The surface-exposed positions can be determined based on the information on the three dimensional structure of antibodies using software suitable for protein modeling. Software which is suitably used for this purpose includes the SYBYL biopolymer module software (Tripos Associates). When the algorithm requires the input size parameter from the user, the “size” of probe for use in computation is generally or preferably set at about 1.4 angstrom or less in radius. Furthermore, a method for determining surface-exposed region and area using PC software is described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; and J. Mol. Model. (1995) 1, 46-53).

[0795] In still another non-limiting embodiment of the present invention, a naive library constructed from antibody genes derived from lymphocytes of healthy persons and consisting of naive sequences, which are unbiased repertoire of antibody sequences, can also be particularly suitably used as a randomized variable region library (Gejima et al. (Human Antibodies (2002) 11, 121-129); and Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)).FcRn

[0796] Unlike Fcγ receptor belonging to the immunoglobulin superfamily, FcRn, human FcRn in particular, is structurally similar to polypeptides of major histocompatibility complex (MHC) class I, exhibiting 22% to 29% sequence identity to class I MHC molecules (Ghetie el al., Immunol. Today (1997) 18 (12): 592-598). FcRn is expressed as a heterodimer consisting of soluble β or light chain (β2 microglobulin) complexed with transmembrane a or heavy chain. Like MHC, FcRn α chain comprises three extracellular domains (α1, α2, and α3) and its short cytoplasmic domain anchors the protein onto the cell surface. α1 and α2 domains interact with the FcRn-binding domain of the antibody Fc region (Raghavan et al., Immunity (1994) 1: 303-315).

[0797] FcRn is expressed in maternal placenta and york sac of mammals, and is involved in mother-to-fetus IgG transfer. In addition, in neonatal small intestine of rodents, where FcRn is expressed, FcRn is involved in transfer of maternal IgG across brush border epithelium from ingested colostrum or milk. FcRn is expressed in a variety of other tissues and endothelial cell systems of various species. FcRn is also expressed in adult human endothelia, muscular blood vessels, and hepatic sinusoidal capillaries. FcRn is believed to play a role in maintaining the plasma IgG concentration by mediating recycling of IgG to serum upon binding to IgG. Typically, binding of FcRn to IgG molecules is strictly pH dependent. The optimal binding is observed in an acidic pH range below 7.0.

[0798] Human FcRn whose precursor is a polypeptide having the signal sequence of SEQ ID NO: 15 (the polypeptide with the signal sequence is shown in SEQ ID NO: 16) forms a complex with human β2-microglobulin in vivo. As shown in the Reference Examples described below, soluble human FcRn complexed with β2-microglobulin is produced by using conventional recombinant expression techniques. FcRn regions of the present invention can be assessed for their binding activity to such a soluble human FcRn complexed with β2-microglobulin. Herein, unless otherwise specified, human FcRn refers to a form capable of binding to an FcRn region of the present invention. Examples include a complex between human FcRn and human β2-microglobulin.FcRn-Binding Domains

[0799] An embodiment of the present invention provides pharmaceutical compositions that induce an immune response to the aforementioned antigen, which comprises as an active ingredient an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an FcRn-binding domain having FcRn-binding activity in a neutral pH range.

[0800] Antigen-binding molecules of the present invention have an FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antigen-binding molecule has FcRn-binding activity in a neutral pH range, and it may be a domain that has activity of directly or indirectly binding to FcRn. Preferred examples of such domains include Fc regions of IgG immunoglobulins, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, anti-FcRn scaffold molecules, and such, which have activity of directly binding to FcRn, or molecules that bind to IgG or albumin, which have activity of indirectly binding to FcRn. For the anti-FcRn scaffold, it is possible to use a domain with any structure of the aforementioned antigen-binding domains characterized by binding to FcRn. In the present invention, a domain having binding activity to FcRn in an acidic pH range and neutral pH range are preferred. Such a domain may be preferably used as it is if it is a domain already having FcRn-binding activity in a neutral pH range. When the domain has no or weak FcRn-binding activity in a neutral pH range, amino acids in the antigen-binding molecule can be modified to impart FcRn-binding activity. Alternatively, FcRn binding activity may be enhanced by altering amino acids in the domain already having FcRn-binding activity in a neutral pH range. Desired amino acid alterations in the FcRn-binding domain can be identified by comparing the FcRn-binding activity in a neutral pH range before and after amino acid alteration.

[0801] Furthermore, in a different embodiment of the present invention, an FcRn binding domain that has FcRn-binding activity under a low calcium ion concentration condition and high calcium ion concentration condition is preferably used. Such a domain may be preferably used as it is if the domain already has FcRn-binding activity under a high calcium ion concentration condition. When the domain has no or weak FcRn-binding activity under a high calcium ion concentration condition, amino acids in the antigen-binding molecule can be modified to impart FcRn-binding activity. Alternatively, FcRn binding activity may be increased by altering amino acids in the domain already having FcRn-binding activity under a high calcium ion concentration condition. Desired amino acid alterations in the FcRn-binding domain can be identified by comparing the FcRn-binding activity under a high calcium ion concentration condition before and after amino acid alteration. FcRn-binding domains may be obtained by methods based on the methods of screening or producing antigen-binding domains having antigen-binding activity that changes depending on calcium ion concentration conditions as mentioned above in the section of “Conditions of ion concentration”. Examples of such FcRn-binding domains include anti-FcRn antibodies, anti-FcRn peptides, anti-FcRn scaffold molecules, and such.

[0802] The preferred human FcRn-binding domain is a region that directly binds to FcRn. Such preferred FcRn-binding domains include, for example, antibody Fc regions. Meanwhile, regions capable of binding to a polypeptide such as albumin or IgG; which has FcRn-binding activity, can indirectly bind to FcRn via albumin, IgG; or such. Therefore, for the FcRn-binding region in the present invention, a region that binds to a polypeptide having FcRn-binding activity may be preferably used. An Fc region contains an amino acid sequence derived from the constant region of an antibody heavy chain. An Fc region is a portion of the antibody heavy chain constant region beginning from the N terminus of the hinge region at the papain cleavage site, which is on the amino acid at approximately position 216 according to EU numbering, and including the hinge, CH2 and CH3 domains.

[0803] The binding activity of an FcRn binding domain of the present invention to FcRn, human FcRn in particular, can be measured by methods known to those skilled in the art, as described in the section “Binding Activity” above. Those skilled in the art can appropriately determine the conditions other than pH. The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be assessed based on the dissociation constant (KD), apparent dissociation constant (KD), dissociation rate (kd), apparent dissociation rate (kd), and such. These can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plot, or flow cytometer may be used.

[0804] When the human FcRn-binding activity of an FcRn-binding domain is measured, conditions other than the pH are not particularly limited, and can be appropriately selected by those skilled in the art. Measurements can be carried out, for example, at 37° C. using MES buffer, as described in WO 2009 / 125825. Alternatively, the FcRn-binding activity of an FcRn-binding domain can be measured by methods known to those skilled in the art, and may be measured by using, for example, Biacore (GE Healthcare) or such. The binding activity of an FcRn-binding domain to FcRn can be assessed by pouring, as an analyte, FcRn, an FcRn-binding domain, or an antigen-binding molecule of the present invention containing the FcRn-binding domain into a chip immobilized with an FcRn-binding domain, an antigen-binding molecule of the present invention containing the FcRn-binding domain, or FcRn.

[0805] The acidic pH range as a condition under which the FcRn-binding domain in the antigen-binding molecule of the present invention has FcRn-binding activity usually refers to pH4.0 to pH6.5. It preferably refers to pH5.5 to pH6.5, and particularly preferably pH5.8 to pH6.0, which is close to the pH in the early-stage endosome in vivo. Furthermore, the neutral pH range as a condition under which the FcRn-binding domain in the antigen-binding molecule of the present invention has FcRn-binding activity usually refers to pH6.7 to pH10.0. The neutral pH range is preferably a range indicated by any pH value within pH7.0 to pH8.0, and is preferably selected from pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and is particularly preferably pH7.4, which is close to the plasma (blood) pH in vivo. When the binding affinity between a human FcRn-binding domain and human FcRn is difficult to evaluate because the binding affinity at pH7.4 is low, pH7.0 can be used instead of pH7.4. For the temperature to be used for the measurement conditions, the binding affinity between the FcRn binding domain and FcRn can be evaluated at any temperature from 10° C. to 50° C. Preferably, a temperature from 15° C. to 40° C. is used to determine the binding affinity between an FcRn-binding domain and human FcRn. More preferably, any temperature from 20° C. to 35° C., such as any temperature selected from 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35° C., is used similarly to determine the binding affinity between an FcRn binding domain and FcRn. In an embodiment of the present invention, 25° C. is a non-limiting example of such temperature.

[0806] According to the Journal of Immunology (2009) 182: 7663-7671, the human FcRn-binding activity of naturally occurring human IgG1 is a KD of 1.7 μM in the acidic pH range (pH6.0), but hardly detectable in the neutral pH range. Therefore, in a preferred embodiment, antigen-binding molecules of the present invention having human FcRn-binding activity in an acidic pH range may be used, which include antigen-binding molecules whose human FcRn-binding activity in an acidic pH range is a KD of 20 μM or stronger, and whose human FcRn-binding activity in a neutral pH range is equivalent to that of naturally-occurring human IgG In a more preferred embodiment, antigen-binding molecules of the present invention including antigen-binding molecules whose human FcRn-binding activity in an acidic pH range is a KD of 2.0 μM or stronger may be used. In an even more preferred embodiment, antigen-binding molecules whose human FcRn-binding activity in an acidic pH range is a KD of 0.5 μM or stronger may be used. The above-mentioned KD values are determined by the method described in the Journal of Immunology (2009) 182: 7663-7671 (antigen-binding molecules are immobilized onto a chip and human FcRn is allowed to flow as an analyte).

[0807] In the present invention, an Fc region having FcRn-binding activity in an acidic pH range is preferred. If such a domain is an Fc region already having FcRn-binding activity in an acidic pH range, it may be used as it is. If the domain has no or weak FcRn-binding activity in an acidic pH range, an Fc region having desired FcRn-binding activity may be obtained by altering amino acids in the antigen-binding molecule. Also, an Fc region having desired or enhanced FcRn-binding activity in an acidic pH range may be suitably obtained by altering amino acids in the Fc region. Amino acid alterations of the Fc region that lead to such desired binding activity may be determined by comparing the FcRn-binding activity in an acidic pH range before and after amino acid alteration. Persons skilled in the art can appropriately perform amino acid alteration using known methods such as overlap extension PCR and site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) similarly to the aforementioned methods used to alter antigen-binding activity.

[0808] An Fc region having FcRn-binding activity in an acidic pH range that is contained in the antigen-binding molecules of the present invention may be obtained by any methods, but specifically, an FcRn-binding domain having FcRn-binding activity or enhanced FcRn-binding activity in an acidic pH range may be obtained by altering amino acids of human IgG immunoglobulin that is used as a starting Fc region. Examples of preferred IgG immunoglobulin Fc regions to be altered include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and their variants). Amino acids at any positions may be altered to other amino acids as long as the Fc region has FcRn-binding activity in an acidic pH range or its human FcRn-binding activity in an acidic range can be enhanced. When an antigen-binding molecule includes the Fc region of human IgG1, it is preferred to include alterations that result in enhancement of FcRn-binding in an acidic pH range as compared to the binding activity of the starting Fc region of human IgG1. Examples of amino acids to which such alterations can be made preferably include, for example, amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and / or 447 according to EU numbering, as described in WO 2000 / 042072. Similarly, examples of amino acids to which such alterations can be made also preferably include amino acids at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and / or 436 according to EU numbering, as described in WO 2002 / 060919. Furthermore, examples of amino acids to which such alterations can be made also include amino acids at positions 250, 314, and 428 according to EU numbering as described in WO 2004 / 092219. In addition, examples of amino acids to which such alterations can be made also preferably include amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434, and / or 436 according to EU numbering as described in WO 2010 / 045193. Alteration of these amino acids enhances the binding of an IgG immunoglobulin Fc region to FcRn in an acidic pH range.

[0809] In the present invention, an Fc region having FcRn-binding activity in a neutral pH range is preferred. If the domain is an Fc region already having FcRn-binding activity in a neutral pH range, it may be used as it is. When the domain has no or weak FcRn-binding activity in a neutral pH range, an Fc region having desired FcRn-binding activity may be obtained by altering the amino acids in the antigen-binding molecule. Also, an Fc region having desired or enhanced FcRn-binding activity in a neutral pH range may be suitably obtained by altering amino acids in the Fc region. Amino acid alterations of the Fc region that lead to such desired binding activity may be determined by comparing the FcRn-binding activity in a neutral pH range before and after amino acid alteration. Persons skilled in the art can suitably perform amino acid alteration using known methods such as overlap extension PCR or site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) similarly to the aforementioned methods used to alter antigen-binding activity.

[0810] An Fc region having FcRn-binding activity in a neutral pH range that is contained in the antigen-binding molecule of the present invention may be obtained by any method, but specifically, an FcRn-binding domain having FcRn-binding activity or enhanced FcRn-binding activity in a neutral pH range may be obtained by amino acid modification of human IgG immunoglobulin used as a starting Fc region. Examples of preferred IgG immunoglobulin Fc regions to be modified include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and their variants). Amino acids at any positions may be altered to other amino acids as long as the Fc region has FcRn-binding activity in a neutral pH range or its human FcRn-binding activity in the neutral range can be enhanced. When an antigen-binding molecule includes the Fc region of human IgG1, it is preferred to include alterations that result in enhancement of FcRn-binding in a neutral pH range compared to the binding activity of the starting Fc region of human IgG1. KD values for FcRn in a neutral pH range are determined as described above by the method described in the Journal of Immunology (2009) 182: 7663-7671 (antigen-binding molecules are immobilized onto a chip and human FcRn is allowed to flow as the analyte).

[0811] Examples of preferred IgG immunoglobulin Fc regions to be altered include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and their variants). Amino acids at any positions may be altered to other amino acids as long as the Fc region has FcRn-binding activity in a neutral pH range or its human FcRn-binding activity in a neutral range can be enhanced. When an antigen-binding molecule includes the Fc region of human IgG1, it is preferred to include alterations that result in enhancement of FcRn-binding in a neutral pH range compared to the binding activity of the starting Fc region of human IgG1. In order to produce Fc regions to which such alterations have been made, various mutations shown in Table 5 were introduced into VH3-IgG1 (SEQ ID NO: 17) and evaluated. Variants (IgG1-F1 to IgG1-F1052) each containing a produced heavy chain and a light chain, L (WT) (SEQ ID NO: 18), were expressed and purified according to the methods described in Reference Example 1.

[0812] Binding between an antibody and human FcRn was analyzed according to the method described in Example 3-3. The binding activity of the variants to human FcRn under neutral conditions (pH7.0) measured using Biacore are shown in Table 5 (Table 5-1 to Table 5-33).

[0813] TABLE 5-1VARIANTKD (M)AMINO ACID ALTERATION POSITIONF18.10E−07N434WF23.20E−06M252Y / S254T / T256EF32.50E−06N434YF45.80E−06N434SF56.80E−06N434AF75.60E−06M252YF84.20E−06M252WF91.40E−07M252Y / S254T / T256E / N434YF106.90E−08M252Y / S254T / T256E / N434WF113.10E−07M252Y / N434YF121.70E−07M252Y / N434WF133.20E−07M252W / N434YF141.80E−07M252W / N434WF194.60E−07P257L / N434YF204.60E−07V308F / N434YF213.00E−08M252Y / V308P / N434YF222.00E−06M428L / N434SF259.20E−09M252Y / S254T / T256E / V308P / N434WF261.00E−06I332VF277.40E−06G237MF291.40E−06I332V / N434YF312.80E−06G237M / V308FF328.00E−07S254T / N434WF332.30E−06S254T / N434YF342.80E−07T256E / N434WF358.40E−07T256E / N434YF363.60E−07S254T / T256E / N434WF371.10E−06S254T / T256E / N434YF381.00E−07M252Y / S254T / N434WF393.00E−07M252Y / S254T / N434YF408.20E−08M252Y / T256E / N434WF411.50E−07M252Y / T256E / N434Y

[0814] TABLE 5-2F421.00E−06M252Y / S254T / T256E / N434AF431.70E−06M252Y / N434AF441.10E−06M252W / N434AF472.40E−07M252Y / T256Q / N434WF483.20E−07M252Y / T256Q / N434YF495.10E−07M252F / T256D / N434WF501.20E−06M252F / T256D / N434YF518.10E−06N434F / Y436HF523.10E−06H433K / N434F / Y436HF531.00E−06I332V / N434WF548.40E−08V308P / N434WF569.40E−07I332V / M428L / N434YF571.10E−05G385D / Q386P / N389SF587.70E−07G385D / Q386P / N389S / N434WF592.40E−06G385D / Q386P / N389S / N434YF601.10E−05G385HF619.70E−07G385H / N434WF621.90E−06G385H / N434YF632.50E−06N434FF645.30E−06N434HF652.90E−07M252Y / S254T / T256E / N434FF664.30E−07M252Y / S254T / T256E / N434HF676.30E−07M252Y / N434FF689.30E−07M252Y / N434HF695.10E−07M428L / N434WF701.50E−06M428L / N434YF718.30E−08M252Y / S254T / T256E / M428L / N434WF722.00E−07M252Y / S254T / T256E / M428L / N434YF731.70E−07M252Y / M428L / N434WF744.60E−07M252Y / M428L / N434YF751.40E−06M252Y / M428L / N434AF761.00E−06M252Y / S254T / T256E / M428L / N434AF779.90E−07T256E / M428L / N434YF787.80E−07S254T / M428L / N434W

[0815] TABLE 5-3F795.90E−06S254T / T256E / N434AF802.70E−06M252Y / T256Q / N434AF811.60E−06M252Y / T256E / N434AF821.10E−06T256Q / N434WF832.60E−06T256Q / N434YF842.80E−07M252W / T256Q / N434WF855.50E−07M252W / T256Q / N434YF861.50E−06S254T / T256Q / N434WF874.30E−06S254T / T256Q / N434YF881.90E−07M252Y / S254T / T256Q / N434WF893.60E−07M252Y / S254T / T256Q / N434YF901.90E−08M252Y / T256E / V308P / N434WF914.80E−08M252Y / V308P / M428L / N434YF921.10E−08M252Y / S254T / T256E / V308P / M428L / N434WF937.40E−07M252W / M428L / N434WF943.70E−07P257L / M428L / N434YF952.60E−07M252Y / S254T / T256E / M428L / N434FF996.20E−07M252Y / T256E / N434HF1011.10E−07M252W / T256Q / P257L / N434YF1034.40E−08P238A / M252Y / V308P / N434YF1043.70E−08M252Y / D265A / V308P / N434YF1057.50E−08M252Y / T307A / V308P / N434YF1063.70E−08M252Y / V303A / V308P / N434YF1073.40E−08M252Y / V308P / D376A / N434YF1084.10E−08M252Y / V305A / V308P / N434YF1093.20E−08M252Y / V308P / Q311A / N434YF1113.20E−08M252Y / V308P / K317A / N434YF1126.40E−08M252Y / V308P / E380A / N434YF1133.20E−08M252Y / V308P / E382A / N434YF1143.80E−08M252Y / V308P / S424A / N434YF1156.60E−06T307A / N434AF1168.70E−06E380A / N434AF1181.40E−05M428LF1195.40E−06T250Q / M428L

[0816] TABLE 5-4F1206.30E−08P257L / V308P / M428L / N434YF1211.50E−08M252Y / T256E / V308P / M428L / N434WF1221.20E−07M252Y / T256E / M428L / N434WF1233.00E−08M252Y / T256E / V308P / N434YF1242.90E−07M252Y / T256E / M428L / N434YF1252.40E−08M252Y / S254T / T256E / V308P / M428L / N434YF1281.70E−07P257L / M428L / N434WF1292.20E−07P257A / M428L / N434YF1313.00E−06P257G / M428L / N434YF1322.10E−07P257I / M428L / N434YF1334.10E−07P257M / M428L / N434YF1342.70E−07P257N / M428L / N434YF1357.50E−07P257S / M428L / N434YF1363.80E−07P257T / M428L / N434YF1374.60E−07P257V / M428L / N434YF1391.50E−08M252W / V308P / N434WF1403.60E−08S239K / M252Y / V308P / N434YF1413.50E−08M252Y / S298G / V308P / N434YF1423.70E−08M252Y / D270F / V308P / N434YF1432.00E−07M252Y / V308A / N434YF1455.30E−08M252Y / V308F / N434YF1472.40E−07M252Y / V308I / N434YF1491.90E−07M252Y / V308L / N434YF1502.00E−07M252Y / V308M / N434YF1522.70E−07M252Y / V308Q / N434YF1541.80E−07M252Y / V308T / N434YF1571.50E−07P257A / V308P / M428L / N434YF1585.90E−08P257T / V308P / M428L / N434YF1594.40E−08P257V / V308P / M428L / N434YF1608.50E−07M252W / M428I / N434YF1621.60E−07M252W / M428Y / N434YF1634.20E−07M252W / M428F / N434YF1643.70E−07P238A / M252W / N434YF1652.90E−07M252W / D265A / N434Y

[0817] TABLE 5-5F1661.50E−07M252W / T307Q / N434YF1672.90E−07M252W / V303A / N434YF1683.20E−07M252W / D376A / N434YF1692.90E−07M252W / V305A / N434YF1701.70E−07M252W / Q311A / N434YF1711.90E−07M252W / D312A / N434YF1722.20E−07M252W / K317A / N434YF1737.70E−07M252W / E380A / N434YF1743.40E−07M252W / E382A / N434YF1752.70E−07M252W / S424A / N434YF1762.90E−07S239K / M252W / N434YF1772.80E−07M252W / S298G / N434YF1782.70E−07M252W / D270F / N434YF1793.10E−07M252W / N325G / N434YF1826.60E−08P257A / M428L / N434WF1832.20E−07P257T / M428L / N434WF1842.70E−07P257V / M428L / N434WF1852.60E−07M252W / I332V / N434YF1883.00E−06P257I / Q311IF1891.90E−07M252Y / T307A / N434YF1901.10E−07M252Y / T307Q / N434YF1911.60E−07P257L / T307A / M428L / N434YF1921.10E−07P257A / T307A / M428L / N434YF1938.50E−08P257T / T307A / M428L / N434YF1941.20E−07P257V / T307A / M428L / N434YF1955.60E−08P257L / T307Q / M428L / N434YF1963.50E−08P257A / T307Q / M428L / N434YF1973.30E−08P257T / T307Q / M428L / N434YF1984.80E−08P257V / T307Q / M428L / N434YF2012.10E−07M252Y / T307D / N434YF2032.40E−07M252Y / T307F / N434YF2042.10E−07M252Y / T307G / N434YF2052.00E−07M252Y / T307H / N434YF2062.30E−07M252Y / T307I / N434Y

[0818] TABLE 5-6F2079.40E−07M252Y / T307K / N434YF2083.90E−07M252Y / T307L / N434YF2091.30E−07M252Y / T307M / N434YF2102.90E−07M252Y / T307N / N434YF2112.40E−07M252Y / T307P / N434YF2126.80E−07M252Y / T307R / N434YF2132.30E−07M252Y / T307S / N434YF2141.70E−07M252Y / T307V / N434YF2159.60E−08M252Y / T307W / N434YF2162.30E−07M252Y / T307Y / N434YF2172.30E−07M252Y / K334L / N434YF2182.60E−07M252Y / G385H / N434YF2192.50E−07M252Y / T289H / N434YF2202.50E−07M252Y / Q311H / N434YF2213.10E−07M252Y / D312H / N434YF2223.40E−07M252Y / N315H / N434YF2232.70E−07M252Y / K360H / N434YF2251.50E−06M252Y / L314R / N434YF2265.40E−07M252Y / L314K / N434YF2271.20E−07M252Y / N286E / N434YF2282.30E−07M252Y / L309E / N434YF2295.10E−07M252Y / R255E / N434YF2302.50E−07M252Y / P387E / N434YF2368.90E−07K248I / M428L / N434YF2372.30E−07M252Y / M428A / N434YF2387.40E−07M252Y / M428D / N434YF2407.20E−07M252Y / M428F / N434YF2411.50E−06M252Y / M428G / N434YF2428.50E−07M252Y / M428H / N434YF2431.80E−07M252Y / M428I / N434YF2441.30E−06M252Y / M428K / N434YF2454.70E−07M252Y / M428N / N434YF2461.10E−06M252Y / M428P / N434YF2474.40E−07M252Y / M428Q / N434Y

[0819] TABLE 5-7F2496.40E−07M252Y / M428S / N434YF2502.90E−07M252Y / M428T / N434YF2511.90E−07M252Y / M428V / N434YF2521.00E−06M252Y / M428W / N434YF2537.10E−07M252Y / M428Y / N434YF2547.50E−08M252W / T307Q / M428Y / N434YF2551.10E−07M252W / Q311A / M428Y / N434YF2565.40E−08M252W / T307Q / Q311A / M428Y / N434YF2575.00E−07M252Y / T307A / M428Y / N434YF2583.20E−07M252Y / T307Q / M428Y / N434YF2592.80E−07M252Y / D270F / N434YF2601.30E−07M252Y / T307A / Q311A / N434YF2618.40E−08M252Y / T307Q / Q311A / N434YF2621.90E−07M252Y / T307A / Q311H / N434YF2631.10E−07M252Y / T307Q / Q311H / N434YF2642.80E−07M252Y / E382A / N434YF2656.80E−07M252Y / E382A / M428Y / N434YF2664.70E−07M252Y / T307A / E382A / M428Y / N434YF2673.20E−07M252Y / T307Q / E382A / M428Y / N434YF2686.30E−07P238A / M252Y / M428F / N434YF2695.20E−07M252Y / V305A / M428F / N434YF2706.60E−07M252Y / N325G / M428F / N434YF2716.90E−07M252Y / D376A / M428F / N434YF2726.80E−07M252Y / E380A / M428F / N434YF2736.50E−07M252Y / E382A / M428F / N434YF2747.60E−07M252Y / E380A / E382A / M428F / N434YF2754.20E−08S239K / M252Y / V308P / E382A / N434YF2764.10E−08M252Y / D270F / V308P / E382A / N434YF2771.30E−07S239K / M252Y / V308P / M428Y / N434YF2783.00E−08M252Y / T307Q / V308P / E382A / N434YF2796.10E−08M252Y / V308P / Q311H / E382A / N434YF2804.10E−08S239K / M252Y / D270F / V308P / N434YF2819.20E−08M252Y / V308P / E382A / M428F / N434YF2822.90E−08M252Y / V308P / E382A / M428L / N434Y

[0820] TABLE 5-8F2831.00E−07M252Y / V308P / E382A / M428Y / N434YF2841.00E−07M252Y / V308P / M428Y / N434YF2859.90E−08M252Y / V308P / M428F / N434YF2861.20E−07S239K / M252Y / V308P / E382A / M428Y / N434YF2871.00E−07M252Y / V308P / E380A / E382A / M428F / N434YF2881.90E−07M252Y / T256E / E382A / N434YF2894.80E−07M252Y / T256E / M428Y / N434YF2904.60E−07M252Y / T256E / E382A / M428Y / N434YF2922.30E−08S239K / M252Y / V308P / E382A / M428I / N434YF2935.30E−08M252Y / V308P / E380A / E382A / M428I / N434YF2941.10E−07S239K / M252Y / V308P / M428F / N434YF2956.80E−07S239K / M252Y / E380A / E382A / M428F / N434YF2964.90E−07M252Y / Q311A / M428Y / N434YF2975.10E−07M252Y / D312A / M428Y / N434YF2984.80E−07M252Y / Q311A / D312A / M428Y / N434YF2999.40E−08S239K / M252Y / V308P / Q311A / M428Y / N434YF3008.30E−08S239K / M252Y / V308P / D312A / M428Y / N434YF3017.20E−08S239K / M252Y / V308P / Q311A / D312A / M428Y / N434YF3021.90E−07M252Y / T256E / T307P / N434YF3036.70E−07M252Y / T307P / M428Y / N434YF3041.60E−08M252W / V308P / M428Y / N434YF3052.70E−08M252Y / T256E / V308P / E382A / N434YF3063.60E−08M252W / V308P / E382A / N434YF3073.60E−08S239K / M252W / V308P / E382A / N434YF3081.90E−08S239K / M252W / V308P / E382A / M428Y / N434YF3109.40E−08S239K / M252W / V308P / E382A / M428I / N434YF3112.80E−08S239K / M252W / V308P / M428F / N434YF3124.50E−07S239K / M252W / E380A / E382A / M428F / N434YF3136.50E−07S239K / M252Y / T307P / M428Y / N434YF3143.20E−07M252Y / T256E / Q311A / D312A / M428Y / N434YF3156.80E−07S239K / M252Y / M428Y / N434YF3167.00E−07S239K / M252Y / D270F / M428Y / N434YF3171.10E−07S239K / M252Y / D270F / V308P / M428Y / N434YF3181.80E−08S239K / M252Y / V308P / M428I / N434Y

[0821] TABLE 5-9F3202.00E−08S239K / M252Y / V308P / N325G / E382A / M428I / N434YF3213.20E−08S239K / M252Y / D270F / V308P / N325G / N434YF3229.20E−08S239K / M252Y / D270F / T307P / V308P / N434YF3232.70E−08S239K / M252Y / T256E / D270F / V308P / N434YF3242.80E−08S239K / M252Y / D270F / T307Q / V308P / N434YF3252.10E−08S239K / M252Y / D270F / T307Q / V308P / Q311A / N434YF3267.50E−08S239K / M252Y / D270F / T307Q / Q311A / N434YF3276.50E−08S239K / M252Y / T256E / D270F / T307Q / Q311A / N434YF3281.90E−08S239K / M252Y / D270F / V308P / M428I / N434YF3291.20E−08S239K / M252Y / D270F / N286E / V308P / N434YF3303.60E−08S239K / M252Y / D270F / V308P / L309E / N434YF3313.00E−08S239K / M252Y / D270F / V308P / P387E / N434YF3337.40E−08S239K / M252Y / D270F / T307Q / L309E / Q311A / N434YF3341.90E−08S239K / M252Y / D270F / V308P / N325G / M428I / N434YF3351.50E−08S239K / M252Y / T256E / D270F / V308P / M428I / N434YF3361.40E−08S239K / M252Y / D270F / T307Q / V308P / Q311A / M428I / N434YF3375.60E−08S239K / M252Y / D270F / T307Q / Q311A / M428I / N434YF3387.70E−09S239K / M252Y / D270F / N286E / V308P / M428I / N434YF3391.90E−08S239K / M252Y / D270F / V308P / L309E / M428I / N434YF3433.20E−08S239K / M252Y / D270F / V308P / M428L / N434YF3443.00E−08S239K / M252Y / V308P / M428L / N434YF3491.50E−07S239K / M252Y / V308P / L309P / M428L / N434YF3501.70E−07S239K / M252Y / V308P / L309R / M428L / N434YF3526.00E−07S239K / M252Y / L309P / M428L / N434YF3531.10E−06S239K / M252Y / L309R / M428L / N434YF3542.80E−08S239K / M252Y / T307Q / V308P / M428L / N434YF3563.40E−08S239K / M252Y / D270F / V308P / L309E / P387E / N434YF3571.60E−08S239K / M252Y / T256E / D270F / V308P / N325G / M428I / N434YF3581.00E−07S239K / M252Y / T307Q / N434YF3594.20E−07P257V / T307Q / M428I / N434YF3601.30E−06P257V / T307Q / M428V / N434YF3625.40E−08P257V / T307Q / N325G / M428L / N434YF3634.10E−08P257V / T307Q / Q311A / M428L / N434YF3643.50E−08P257V / T307Q / Q311A / N325G / M428L / N434Y

[0822] TABLE 5-10F3655.10E−08P257V / V305A / T307Q / M428L / N434YF3671.50E−08S239K / M252Y / E258H / D270F / T307Q / V308P / Q311A / N434YF3682.00E−08S239K / M252Y / D270F / V308P / N325G / E382A / M428I / N434YF3697.50E−08M252Y / P257V / T307Q / M428I / N434YF3721.30E−08S239K / M252W / V308P / M428Y / N434YF3731.10E−08S239K / M252W / V308P / Q311A / M428Y / N434YF3741.20E−08S239K / M252W / T256E / V308P / M428Y / N434YF3755.50E−09S239K / M252W / N286E / V308P / M428Y / N434YF3769.60E−09S239K / M252Y / T256E / D270F / N286E / V308P / N434YF3771.30E−07S239K / M252W / T307P / M428Y / N434YF3799.00E−09S239K / M252W / T256E / V308P / Q311A / M428Y / N434YF3805.60E−09S239K / M252W / T256E / N286E / V308P / M428Y / N434YF3811.10E−07P257V / T307A / Q311A / M428L / N434YF3828.70E−08P257V / V305A / T307A / M428L / N434YF3863.20E−08M252Y / V308P / L309E / N434YF3871.50E−07M252Y / V308P / L309D / N434YF3887.00E−08M252Y / V308P / L309A / N434YF3891.70E−08M252W / V308P / L309E / M428Y / N434YF3906.80E−08M252W / V308P / L309D / M428Y / N434YF3913.60E−08M252W / V308P / L309A / M428Y / N434YF3926.90E−09S239K / M252Y / N286E / V308P / M428I / N434YF3931.20E−08S239K / M252Y / N286E / V308P / N434YF3945.30E−08S239K / M252Y / T307Q / Q311A / M428I / N434YF3952.40E−08S239K / M252Y / T256E / V308P / N434YF3962.00E−08S239K / M252Y / D270F / N286E / T307Q / Q311A / M428I / N434YF3974.50E−08S239K / M252Y / D270F / T307Q / Q311A / P387E / M428I / N434YF3984.40E−09S239K / M252Y / D270F / N286E / T307Q / V308P / Q311A / M428I / N434YF3996.50E−09S239K / M252Y / D270F / N286E / T307Q / V308P / M428I / N434YF4006.10E−09S239K / M252Y / D270F / N286E / V308P / Q311A / M428I / N434YF4016.90E−09S239K / M252Y / D270F / N286E / V308P / P387E / M428I / N434YF4022.30E−08P257V / T307Q / M428L / N434WF4035.10E−08P257V / T307A / M428L / N434WF4049.40E−08P257A / T307Q / L309P / M428L / N434YF4051.70E−07P257V / T307Q / L309P / M428L / N434Y

[0823] TABLE 5-11F4061.50E−07P257A / T307Q / L309R / M428L / N434YF4071.60E−07P257V / T307Q / L309R / M428L / N434YF4082.50E−07P257V / N286E / M428L / N434YF4092.00E−07P257V / P387E / M428L / N434YF4102.20E−07P257V / T307H / M428L / N434YF4111.30E−07P257V / T307N / M428L / N434YF4128.80E−08P257V / T307G / M428L / N434YF4131.20E−07P257V / T307P / M428L / N434YF4141.10E−07P257V / T307S / M428L / N434YF4155.60E−08P257V / N286E / T307A / M428L / N434YF4169.40E−08P257V / T307A / P387E / M428L / N434YF4186.20E−07S239K / M252Y / T307P / N325G / M428Y / N434YF4191.60E−07M252Y / T307A / Q311H / K360H / N434YF4201.50E−07M252Y / T307A / Q311H / P387E / N434YF4211.30E−07M252Y / T307A / Q311H / M428A / N434YF4221.80E−07M252Y / T307A / Q311H / E382A / N434YF4238.40E−08M252Y / T307W / Q311H / N434YF4249.40E−08S239K / P257A / V308P / M428L / N434YF4258.00E−08P257A / V308P / L309E / M428L / N434YF4268.40E−08P257V / T307Q / N434YF4271.10E−07M252Y / P257V / T307Q / M428V / N434YF4288.00E−08M252Y / P257V / T307Q / M428L / N434YF4293.70E−08M252Y / P257V / T307Q / N434YF4308.10E−08M252Y / P257V / T307Q / M428Y / N434YF4316.50E−08M252Y / P257V / T307Q / M428F / N434YF4329.20E−07P257V / T307Q / Q311A / N325G / M428V / N434YF4336.00E−08P257V / T307Q / Q311A / N325G / N434YF4342.00E−08P257V / T307Q / Q311A / N325G / M428Y / N434YF4352.50E−08P257V / T307Q / Q311A / N325G / M428F / N434YF4362.50E−07P257A / T307Q / M428V / N434YF4375.70E−08P257A / T307Q / N434YF4383.60E−08P257A / T307Q / M428Y / N434YF4394.00E−08P257A / T307Q / M428F / N434YF4401.50E−08P257V / N286E / T307Q / Q311A / N325G / M428L / N434Y

[0824] TABLE 5-12F4411.80E−07P257A / Q311A / M428L / N434YF4422.00E−07P257A / Q311H / M428L / N434YF4435.50E−08P257A / T307Q / Q311A / M428L / N434YF4441.40E−07P257A / T307A / Q311A / M428L / N434YF4456.20E−08P257A / T307Q / Q311H / M428L / N434YF4461.10E−07P257A / T307A / Q311H / M428L / N434YF4471.40E−08P257A / N286E / T307Q / M428L / N434YF4485.30E−08P257A / N286E / T307A / M428L / N434YF4495.70E−07S239K / M252Y / D270F / T307P / N325G / M428Y / N434YF4505.20E−07S239K / M252Y / T307P / L309E / N325G / M428Y / N434YF4511.00E−07P257S / T307A / M428L / N434YF4521.40E−07P257M / T307A / M428L / N434YF4537.80E−08P257N / T307A / M428L / N434YF4549.60E−08P257I / T307A / M428L / N434YF4552.70E−08P257V / T307Q / M428Y / N434YF4563.40E−08P257V / T307Q / M428F / N434YF4574.00E−08S239K / P257V / V308P / M428L / N434YF4581.50E−08P257V / T307Q / V308P / N325G / M428L / N434YF4591.30E−08P257V / T307Q / V308P / Q311A / N325G / M428L / N434YF4604.70E−08P257V / T307A / V308P / N325G / M428L / N434YF4628.50E−08P257A / V308P / N325G / M428L / N434YF4631.30E−07P257A / T307A / V308P / M428L / N434YF4645.50E−08P257A / T307Q / V308P / M428L / N434YF4652.10E−08P257V / N286E / T307Q / N325G / M428L / N434YF4663.50E−07T256E / P257V / N434YF4675.70E−07T256E / P257T / N434YF4685.70E−08S239K / P257T / V308P / M428L / N434YF4695.60E−08P257T / V308P / N325G / M428L / N434YF4705.40E−08T256E / P257T / V308P / N325G / M428L / N434YF4716.60E−08P257T / V308P / N325G / E382A / M428L / N434YF4725.40E−08P257T / V308P / N325G / P387E / M428L / N434YF4734.50E−07P257T / V308P / L309P / N325G / M428L / N434YF4743.50E−07P257T / V308P / L309R / N325G / M428L / N434YF4754.30E−08T256E / P257V / T307Q / M428L / N434Y

[0825] TABLE 5-13F4765.50E−08P257V / T307Q / E382A / M428L / N434YF4774.30E−08P257V / T307Q / P387E / M428L / N434YF4803.90E−08P257L / V308P / N434YF4815.60E−08P257T / T307Q / N434YF4827.00E−08P257V / T307Q / N325G / N434YF4835.70E−08P257V / T307Q / Q311A / N434YF4846.20E−08P257V / V305A / T307Q / N434YF4859.70E−08P257V / N286E / T307A / N434YF4863.40E−07P257V / T307Q / L309R / Q311H / M428L / N434YF4883.50E−08P257V / V308P / N325G / M428L / N434YF4907.50E−08S239K / P257V / V308P / Q311H / M428L / N434YF4929.80E−08P257V / V305A / T307A / N325G / M428L / N434YF4934.90E−07S239K / D270F / T307P / N325G / M428Y / N434YF4973.10E−06P257T / T307A / M428V / N434YF4981.30E−06P257A / M428V / N434YF4995.20E−07P257A / T307A / M428V / N434YF5004.30E−08P257S / T307Q / M428L / N434YF5061.90E−07P257V / N297A / T307Q / M428L / N434YF5075.10E−08P257V / N286A / T307Q / M428L / N434YF5081.10E−07P257V / T307Q / N315A / M428L / N434YF5095.80E−08P257V / T307Q / N384A / M428L / N434YF5105.30E−08P257V / T307Q / N389A / M428L / N434YF5114.20E−07P257V / N434YF5125.80E−07P257T / N434YF5173.10E−07P257V / N286E / N434YF5184.20E−07P257T / N286E / N434YF5192.60E−08P257V / N286E / T307Q / N434YF5211.10E−08P257V / N286E / T307Q / M428Y / N434YF5232.60E−08P257V / V305A / T307Q / M428Y / N434YF5261.90E−08P257T / T307Q / M428Y / N434YF5279.40E−09P257V / T307Q / V308P / N325G / M428Y / N434YF5292.50E−08P257T / T307Q / M428F / N434YF5331.20E−08P257A / N286E / T307Q / M428F / N434YF5341.20E−08P257A / N286E / T307Q / M428Y / N434Y

[0826] TABLE 5-14F5353.90E−08T250A / P257V / T307Q / M428L / N434YF5389.90E−08T250F / P257V / T307Q / M428L / N434YF5416.00E−08T250I / P257V / T307Q / M428L / N434YF5443.10E−08T250M / P257V / T307Q / M428L / N434YF5495.40E−08T250S / P257V / T307Q / M428L / N434YF5505.90E−08T250V / P257V / T307Q / M428L / N434YF5511.20E−07T250W / P257V / T307Q / M428L / N434YF5521.10E−07T250Y / P257V / T307Q / M428L / N434YF5531.70E−07M252Y / Q311A / N434YF5542.80E−08S239K / M252Y / S254T / V308P / N434YF5561.50E−06M252Y / T307Q / Q311AF5598.00E−08M252Y / S254T / N286E / N434YF5602.80E−08M252Y / S254T / V308P / N434YF5611.40E−07M252Y / S254T / T307A / N434YF5628.30E−08M252Y / S254T / T307Q / N434YF5631.30E−07M252Y / S254T / Q311A / N434YF5641.90E−07M252Y / S254T / Q311H / N434YF5659.20E−08M252Y / S254T / T307A / Q311A / N434YF5666.10E−08M252Y / S254T / T307Q / Q311A / N434YF5672.20E−07M252Y / S254T / M428I / N434YF5681.10E−07M252Y / T256E / T307A / Q311H / N434YF5692.00E−07M252Y / T256Q / T307A / Q311H / N434YF5701.30E−07M252Y / S254T / T307A / Q311H / N434YF5718.10E−08M252Y / N286E / T307A / Q311H / N434YF5721.00E−07M252Y / T307A / Q311H / M428I / N434YF5761.60E−06M252Y / T256E / T307Q / Q311HF5771.30E−06M252Y / N286E / T307A / Q311AF5785.70E−07M252Y / N286E / T307Q / Q311AF5808.60E−07M252Y / N286E / T307Q / Q311HF5817.20E−08M252Y / T256E / N286E / N434YF5827.50E−07S239K / M252Y / V308PF5837.80E−07S239K / M252Y / V308P / E382AF5846.30E−07S239K / M252Y / T256E / V308PF5852.90E−07S239K / M252Y / N286E / V308P

[0827] TABLE 5-15F5861.40E−07S239K / M252Y / N286E / V308P / M428IF5871.90E−07M252Y / N286E / M428L / N434YF5922.00E−07M252Y / S254T / E382A / N434YF5933.10E−08S239K / M252Y / S254T / V308P / M428I / N434YF5941.60E−08S239K / M252Y / T256E / V308P / M428I / N434YF5951.80E−07S239K / M252Y / M428I / N434YF5964.00E−07M252Y / D312A / E382A / M428Y / N434YF5972.20E−07M252Y / E382A / P387E / N434YF5981.40E−07M252Y / D312A / P387E / N434YF5995.20E−07M252Y / P387E / M428Y / N434YF6002.80E−07M252Y / T256Q / E382A / N434YF6019.60E−09M252Y / N286E / V308P / N434YF608G236A / S239D / I332EF6112.80E−07M252Y / V305T / T307P / V308I / L309A / N434YF6123.60E−07M252Y / T307P / V308I / L309A / N434YF613S239D / A330L / I332EF616S239D / K326D / L328YF6177.40E−07S239K / N434WF6186.40E−07S239K / V308F / N434YF6193.10E−07S239K / M252Y / N434YF6202.10E−07S239K / M252Y / S254T / N434YF6211.50E−07S239K / M252Y / T307A / Q311H / N434YF6223.50E−07S239K / M252Y / T256Q / N434YF6231.80E−07S239K / M252W / N434WF6241.40E−08S239K / P257A / N286E / T307Q / M428L / N434YF6257.60E−08S239K / P257A / T307Q / M428L / N434YF6261.30E−06V308PF6293.90E−08M252Y / V279L / V308P / N434YF6303.70E−08S239K / M252Y / V279L / V308P / N434YF6332.40E−08M252Y / V282D / V308P / N434YF6343.20E−08S239K / M252Y / V282D / V308P / N434YF6354.50E−08M252Y / V284K / V308P / N434YF6364.80E−08S239K / M252Y / V284K / V308P / N434YF6371.50E−07M252Y / K288S / V308P / N434Y

[0828] TABLE 5-16F6381.40E−07S239K / M252Y / K288S / V308P / N434YF6392.70E−08M252Y / V308P / G385R / N434YF6403.60E−08S239K / M252Y / V308P / G385R / N434YF6413.00E−08M252Y / V308P / Q386K / N434YF6423.00E−08S239K / M252Y / V308P / Q386K / N434YF6433.20E−08L235G / G236R / S239K / M252Y / V308P / N434YF6443.00E−08G236R / S239K / M252Y / V308P / N434YF6453.30E−08S239K / M252Y / V308P / L328R / N434YF6463.80E−08S239K / M252Y / N297A / V308P / N434YF6472.90E−08P238D / M252Y / V308P / N434YF648P238DF6491.20E−07S239K / M252Y / N286E / N434YF6501.70E−07S239K / M252Y / T256E / N434YF6511.80E−07S239K / M252Y / Q311A / N434YF6522.40E−07P238D / M252Y / N434YF6543.20E−08L235K / S239K / M252Y / V308P / N434YF6553.40E−08L235R / S239K / M252Y / V308P / N434YF6563.30E−08G237K / S239K / M252Y / V308P / N434YF6573.20E−08G237R / S239K / M252Y / V308P / N434YF6583.20E−08P238K / S239K / M252Y / V308P / N434YF6593.00E−08P238R / S239K / M252Y / V308P / N434YF6603.10E−08S239K / M252Y / V308P / P329K / N434YF6613.40E−08S239K / M252Y / V308P / P329R / N434YF6636.40E−09S239K / M252Y / N286E / T307Q / V308P / Q311A / N434YF6643.90E−08M252Y / N286A / V308P / N434YF6652.00E−08M252Y / N286D / V308P / N434YF6662.10E−08M252Y / N286F / V308P / N434YF6673.00E−08M252Y / N286G / V308P / N434YF6684.00E−08M252Y / N286H / V308P / N434YF6693.50E−08M252Y / N286I / V308P / N434YF6702.10E−07M252Y / N286K / V308P / N434YF6712.20E−08M252Y / N286L / V308P / N434YF6722.40E−08M252Y / N286M / V308P / N434YF6732.30E−08M252Y / N286P / V308P / N434Y

[0829] TABLE 5-17F6743.20E−08M252Y / N286Q / V308P / N434YF6755.10E−08M252Y / N286R / V308P / N434YF6763.20E−08M252Y / N286S / V308P / N434YF6774.70E−08M252Y / N286T / V308P / N434YF6783.30E−08M252Y / N286V / V308P / N434YF6791.70E−08M252Y / N286W / V308P / N434YF6801.50E−08M252Y / N286Y / V308P / N434YF6814.90E−08M252Y / K288A / V308P / N434YF6828.20E−08M252Y / K288D / V308P / N434YF6835.00E−08M252Y / K288E / V308P / N434YF6845.10E−08M252Y / K288F / V308P / N434YF6855.30E−08M252Y / K288G / V308P / N434YF6864.60E−08M252Y / K288H / V308P / N434YF6874.90E−08M252Y / K288I / V308P / N434YF6882.80E−08M252Y / K288L / V308P / N434YF6894.10E−08M252Y / K288M / V308P / N434YF6901.00E−07M252Y / K288N / V308P / N434YF6913.20E−07M252Y / K288P / V308P / N434YF6923.90E−08M252Y / K288Q / V308P / N434YF6933.60E−08M252Y / K288R / V308P / N434YF6944.70E−08M252Y / K288V / V308P / N434YF6954.00E−08M252Y / K288W / V308P / N434YF6964.40E−08M252Y / K288Y / V308P / N434YF6973.10E−08S239K / M252Y / V308P / N325G / N434YF6982.20E−08M252Y / N286E / T307Q / Q311A / N434YF6992.30E−08S239K / M252Y / N286E / T307Q / Q311A / N434YF7005.20E−08M252Y / V308P / L328E / N434YF7057.10E−09M252Y / N286E / V308P / M428I / N434YF7061.80E−08M252Y / N286E / T307Q / Q311A / M428I / N434YF7075.90E−09M252Y / N286E / T307Q / V308P / Q311A / N434YF7084.10E−09M252Y / N286E / T307Q / V308P / Q311A / M428I / N434YF7092.00E−08S239K / M252Y / N286E / T307Q / Q311A / M428I / N434YF7101.50E−08P238D / M252Y / N286E / T307Q / Q311A / M428I / N434YF7116.50E−08S239K / M252Y / T307Q / Q311A / N434Y

[0830] TABLE 5-18F7126.00E−08P238D / M252Y / T307Q / Q311A / N434YF7132.00E−08P238D / M252Y / N286E / T307Q / Q311A / N434YF7142.30E−07P238D / M252Y / N325S / N434YF7152.30E−07P238D / M252Y / N325M / N434YF7162.70E−07P238D / M252Y / N325L / N434YF7172.60E−07P238D / M252Y / N325I / N434YF7182.80E−07P238D / M252Y / Q295M / N434YF7197.40E−08P238D / M252Y / N325G / N434YF7202.40E−08M252Y / T307Q / V308P / Q311A / N434YF7211.50E−08M252Y / T307Q / V308P / Q311A / M428I / N434YF7222.70E−07P238D / M252Y / A327G / N434YF7232.80E−07P238D / M252Y / L328D / N434YF7242.50E−07P238D / M252Y / L328E / N434YF7254.20E−08L235K / G237R / S239K / M252Y / V308P / N434YF7263.70E−08L235K / P238K / S239K / M252Y / V308P / N434YF7299.20E−07T307A / Q311A / N434YF7306.00E−07T307Q / Q311A / N434YF7318.50E−07T307A / Q311H / N434YF7326.80E−07T307Q / Q311H / N434YF7333.20E−07M252Y / L328E / N434YF7343.10E−07G236D / M252Y / L328E / N434YF7363.10E−07M252Y / S267M / L328E / N434YF7373.10E−07M252Y / S267L / L328E / N434YF7383.50E−07P238D / M252Y / T307P / N434YF7392.20E−07M252Y / T307P / Q311A / N434YF7402.90E−07M252Y / T307P / Q311H / N434YF7413.10E−07P238D / T250A / M252Y / N434YF7449.90E−07P238D / T250F / M252Y / N434YF7456.60E−07P238D / T250G / M252Y / N434YF7466.00E−07P238D / T250H / M252Y / N434YF7472.80E−07P238D / T250I / M252Y / N434YF7495.10E−07P238D / T250L / M252Y / N434YF7503.00E−07P238D / T250M / M252Y / N434YF7515.30E−07P238D / T250N / M252Y / N434Y

[0831] TABLE 5-19F7531.80E−07P238D / T250Q / M252Y / N434YF7553.50E−07P238D / T250S / M252Y / N434YF7563.70E−07P238D / T250V / M252Y / N434YF7571.20E−06P238D / T250W / M252Y / N434YF7581.40E−06P238D / T250Y / M252Y / N434YF759L235K / S239KF760L235R / S239KF7611.10E−06P238D / N434YF7623.60E−08L235K / S239K / M252Y / N286E / T307Q / Q311A / N434YF7633.50E−08L235R / S239K / M252Y / N286E / T307Q / Q311A / N434YF7646.30E−07P238D / T307Q / Q311A / N434YF7658.50E−08P238D / M252Y / T307Q / L309E / Q311A / N434YF7666.00E−07T307A / L309E / Q311A / N434YF7674.30E−07T307Q / L309E / Q311A / N434YF7686.40E−07T307A / L309E / Q311H / N434YF7694.60E−07T307Q / L309E / Q311H / N434YF7703.00E−07M252Y / T256A / N434YF7714.00E−07M252Y / E272A / N434YF7723.80E−07M252Y / K274A / N434YF7733.90E−07M252Y / V282A / N434YF7744.00E−07M252Y / N286A / N434YF7756.20E−07M252Y / K338A / N434YF7763.90E−07M252Y / K340A / N434YF7773.90E−07M252Y / E345A / N434YF7793.90E−07M252Y / N361A / N434YF7803.90E−07M252Y / Q362A / N434YF7813.70E−07M252Y / S375A / N434YF7823.50E−07M252Y / Y391A / N434YF7834.00E−07M252Y / D413A / N434YF7845.00E−07M252Y / L309A / N434YF7857.40E−07M252Y / L309H / N434YF7862.80E−08M252Y / S254T / N286E / T307Q / Q311A / N434YF7878.80E−08M252Y / S254T / T307Q / L309E / Q311A / N434YF7884.10E−07M252Y / N315A / N434Y

[0832] TABLE 5-20F7891.50E−07M252Y / N315D / N434YF7902.70E−07M252Y / N315E / N434YF7914.40E−07M252Y / N315F / N434YF7924.40E−07M252Y / N315G / N434YF7933.30E−07M252Y / N315I / N434YF7944.10E−07M252Y / N315K / N434YF7953.10E−07M252Y / N315L / N434YF7963.40E−07M252Y / N315M / N434YF7983.50E−07M252Y / N315Q / N434YF7994.10E−07M252Y / N315R / N434YF8003.80E−07M252Y / N315S / N434YF8014.40E−07M252Y / N315T / N434YF8023.30E−07M252Y / N315V / N434YF8033.60E−07M252Y / N315W / N434YF8044.00E−07M252Y / N315Y / N434YF8053.00E−07M252Y / N325A / N434YF8063.10E−07M252Y / N384A / N434YF8073.20E−07M252Y / N389A / N434YF8083.20E−07M252Y / N389A / N390A / N434YF8092.20E−07M252Y / S254T / T256S / N434YF8102.20E−07M252Y / A378V / N434YF8114.90E−07M252Y / E380S / N434YF8122.70E−07M252Y / E382V / N434YF8132.80E−07M252Y / S424E / N434YF8141.20E−07M252Y / N434Y / Y436IF8155.50E−07M252Y / N434Y / T437RF8163.60E−07P238D / T250V / M252Y / T307P / N434YF8179.80E−08P238D / T250V / M252Y / T307Q / Q311A / N434YF8191.40E−07P238D / M252Y / N286E / N434YF8203.40E−07L235K / S239K / M252Y / N434YF8213.10E−07L235R / S239K / M252Y / N434YF8221.10E−06P238D / T250Y / M252Y / W313Y / N434YF8231.10E−06P238D / T250Y / M252Y / W313F / N434YF8282.50E−06P238D / T250V / M252Y / I253V / N434Y

[0833] TABLE 5-21F8311.60E−06P238D / T250V / M252Y / R255A / N434YF8322.60E−06P238D / T250V / M252Y / R255D / N434YF8338.00E−07P238D / T250V / M252Y / R255E / N434YF8348.10E−07P238D / T250V / M252Y / R255F / N434YF8365.00E−07P238D / T250V / M252Y / R255H / N434YF8375.60E−07P238D / T250V / M252Y / R255I / N434YF8384.30E−07P238D / T250V / M252Y / R255K / N434YF8393.40E−07P238D / T250V / M252Y / R255L / N434YF8404.20E−07P238D / T250V / M252Y / R255M / N434YF8411.10E−06P238D / T250V / M252Y / R255N / N434YF8436.60E−07P238D / T250V / M252Y / R255Q / N434YF8441.30E−06P238D / T250V / M252Y / R255S / N434YF8473.40E−07P238D / T250V / M252Y / R255W / N434YF8488.30E−07P238D / T250V / M252Y / R255Y / N434YF8493.30E−07M252Y / D280A / N434YF8502.90E−07M252Y / D280E / N434YF8523.30E−07M252Y / D280G / N434YF8533.20E−07M252Y / D280H / N434YF8553.20E−07M252Y / D280K / N434YF8583.20E−07M252Y / D280N / N434YF8603.30E−07M252Y / D280Q / N434YF8613.20E−07M252Y / D280R / N434YF8623.00E−07M252Y / D280S / N434YF8632.70E−07M252Y / D280T / N434YF8672.80E−07M252Y / N384A / N389A / N434YF8682.00E−08G236A / S239D / M252Y / N286E / T307Q / Q311A / N434YF869G236A / S239DF8707.30E−08L235K / S239K / M252Y / T307Q / Q311A / N434YF8717.10E−08L235R / S239K / M252Y / T307Q / Q311A / N434YF8721.30E−07L235K / S239K / M252Y / N286E / N434YF8731.20E−07L235R / S239K / M252Y / N286E / N434YF8754.80E−07M252Y / N434Y / Y436AF8778.30E−07M252Y / N434Y / Y436EF8781.90E−07M252Y / N434Y / Y436F

[0834] TABLE 5-22F8799.20E−07M252Y / N434Y / Y436GF8803.90E−07M252Y / N434Y / Y436HF8813.10E−07M252Y / N434Y / Y436KF8821.30E−07M252Y / N434Y / Y436LF8832.10E−07M252Y / N434Y / Y436MF8844.00E−07M252Y / N434Y / Y436NF8884.80E−07M252Y / N434Y / Y436SF8892.20E−07M252Y / N434Y / Y436TF8901.10E−07M252Y / N434Y / Y436VF8911.70E−07M252Y / N434Y / Y436WF8927.10E−08M252Y / S254T / N434Y / Y436IF8939.80E−08L235K / S239K / M252Y / N434Y / Y436IF8949.20E−08L235R / S239K / M252Y / N434Y / Y436IF8952.10E−08L235K / S239K / M252Y / N286E / T307Q / Q311A / N315E / N434YF8962.00E−08L235R / S239K / M252Y / N286E / T307Q / Q311A / N315E / N434YF8979.70E−08M252Y / N315D / N384A / N389A / N434YF8981.70E−07M252Y / N315E / N384A / N389A / N434YF8991.10E−07M252Y / N315D / G316A / N434YF9001.70E−07M252Y / N315D / G316D / N434YF9011.30E−07M252Y / N315D / G316E / N434YF9022.20E−07M252Y / N315D / G316F / N434YF9032.30E−07M252Y / N315D / G316H / N434YF9041.00E−07M252Y / N315D / G316I / N434YF9051.30E−07M252Y / N315D / G316K / N434YF9061.50E−07M252Y / N315D / G316L / N434YF9071.30E−07M252Y / N315D / G316M / N434YF9081.50E−07M252Y / N315D / G316N / N434YF9091.30E−07M252Y / N315D / G316P / N434YF9101.40E−07M252Y / N315D / G316Q / N434YF9111.30E−07M252Y / N315D / G316R / N434YF9121.20E−07M252Y / N315D / G316S / N434YF9131.10E−07M252Y / N315D / G316T / N434YF9141.50E−07M252Y / N315D / G316V / N434YF9152.30E−07M252Y / N315D / G316W / N434Y

[0835] TABLE 5-23F9172.50E−07M252Y / N286S / N434YF9182.80E−07M252Y / D280E / N384A / N389A / N434YF9193.30E−07M252Y / D280G / N384A / N389A / N434YF9202.50E−07M252Y / N286S / N384A / N389A / N434YF9211.20E−07M252Y / N286E / N384A / N389A / N434YF9225.90E−08L235K / S239K / M252Y / N286E / N434Y / Y436IF9236.00E−08L235R / S239K / M252Y / N286E / N434Y / Y436IF9243.40E−08L235K / S239K / M252Y / T307Q / Q311A / N434Y / Y436IF9253.20E−08L235R / S239K / M252Y / T307Q / Q311A / N434Y / Y436IF9261.10E−07L235K / S239K / M252Y / S254T / N434Y / Y436IF9271.00E−07L235R / S239K / M252Y / S254T / N434Y / Y436IF9282.90E−08M252Y / T307Q / Q311A / N434Y / Y436IF9292.90E−08M252Y / S254T / T307Q / Q311A / N434Y / Y436IF9301.40E−07P238D / T250V / M252Y / N286E / N434YF9311.20E−07P238D / T250V / M252Y / N434Y / Y436IF9323.20E−07T250V / M252Y / N434YF9333.00E−07L234R / P238D / T250V / M252Y / N434YF9343.10E−07G236K / P238D / T250V / M252Y / N434YF9353.20E−07G237K / P238D / T250V / M252Y / N434YF9363.20E−07G237R / P238D / T250V / M252Y / N434YF9373.10E−07P238D / S239K / T250V / M252Y / N434YF9381.60E−07L235K / S239K / M252Y / N434Y / Y436VF9391.50E−07L235R / S239K / M252Y / N434Y / Y436VF9401.50E−07P238D / T250V / M252Y / N434Y / Y436VF9411.20E−08M252Y / N286E / T307Q / Q311A / N434Y / Y436VF9424.20E−08L235K / S239K / M252Y / T307Q / Q311A / N434Y / Y436VF9434.00E−08L235R / S239K / M252Y / T307Q / Q311A / N434Y / Y436VF9441.70E−07T250V / M252Y / N434Y / Y436VF9451.70E−08T250V / M252Y / V308P / N434Y / Y436VF9464.30E−08T250V / M252Y / T307Q / Q311A / N434Y / Y436VF9471.10E−08T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF9545.30E−07M252Y / N434Y / H435K / Y436VF9577.70E−07M252Y / N434Y / H435N / Y436VF9608.00E−07M252Y / N434Y / H435R / Y436V

[0836] TABLE 5-24F9663.10E−07M252Y / S254A / N434YF9702.50E−06M252Y / S254G / N434YF9712.60E−06M252Y / S254H / N434YF9722.60E−07M252Y / S254I / N434YF9781.30E−06M252Y / S254Q / N434YF9801.80E−07M252Y / S254V / N434YF9874.00E−08P238D / T250V / M252Y / T307Q / Q311A / N434Y / Y436VF9886.90E−08P238D / T250V / M252Y / N286E / N434Y / Y436VF9891.40E−08L235R / S239K / M252Y / V308P / N434Y / Y436VF9909.40E−09L235R / S239K / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF9911.30E−08L235R / S239K / M252Y / N286E / T307Q / Q311A / N434Y / Y436VF9925.10E−08L235R / S239K / M252Y / T307Q / Q311A / M428I / N434Y / Y436VF9933.80E−08M252Y / T307Q / Q311A / N434Y / Y436VF9942.80E−07M252Y / N325G / N434YF9952.90E−07L235R / P238D / S239K / M252Y / N434YF9961.30E−07L235R / P238D / S239K / M252Y / N434Y / Y436VF9973.80E−07K248I / T250V / M252Y / N434Y / Y436VF9988.50E−07K248Y / T250V / M252Y / N434Y / Y436VF9992.10E−07T250V / M252Y / E258H / N434Y / Y436VF1005N325GF10081.70E−07L235R / S239K / T250V / M252Y / N434Y / Y436VF10091.20E−08L235R / S239K / T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF10101.90E−07L235R / S239K / M252Y / T307A / Q311H / N434YF10114.50E−08T250V / M252Y / V308P / N434YF10124.70E−08L235R / S239K / T250V / M252Y / V308P / N434YF10133.00E−08T250V / M252Y / T307Q / V308P / Q311A / N434YF10143.20E−08L235R / S239K / T250V / M252Y / T307Q / V308P / Q311A / N434YF10152.20E−08L235R / S239K / M252Y / T307Q / V308P / Q311A / N434YF10163.80E−09T250V / M252Y / N286E / T307Q / V308P / Q311A / N434Y / Y436VF10174.20E−09L235R / S239K / T250V / M252Y / N286E / T307Q / V308P / Q311A / N434Y / Y436VF10183.20E−09L235R / S239K / M252Y / N286E / T307Q / V308P / Q311A / N434Y / Y436VF10193.40E−07P238D / T250V / M252Y / N325G / N434YF10208.50E−08P238D / T250V / M252Y / T307Q / Q311A / N325G / N434Y

[0837] TABLE 5-25F10213.30E−07P238D / T250V / M252Y / N325A / N434YF1022K326D / L328YF10234.40E−08S239D / T250V / M252Y / T307Q / Q311A / N434Y / Y436VF10244.00E−08T250V / M252Y / T307Q / Q311A / K326D / L328Y / N434Y / Y436VF10253.60E−08S239D / T250V / M252Y / T307Q / Q311A / K326D / L328Y / N434Y / Y436VF10268.40E−08M252Y / T307A / Q311H / N434Y / Y436VF10278.60E−08L235R / S239K / M252Y / T307A / Q311H / N434Y / Y436VF10284.60E−08G236A / S239D / T250V / M252Y / T307Q / Q311A / N434Y / Y436VF10295.10E−08T250V / M252Y / T307Q / Q311A / I332E / N434Y / Y436VF1030I332EF10315.30E−08G236A / S239D / T250V / M252Y / T307Q / Q311A / I332E / N434Y / Y436VF10324.30E−08P238D / T250V / M252Y / T307Q / Q311A / N325G / N434Y / Y436VF10331.00E−06P238D / N434WF10341.50E−08L235K / S239K / M252Y / V308P / N434Y / Y436VF10351.00E−08L235K / S239K / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF10361.40E−08L235K / S239K / M252Y / N286E / T307Q / Q311A / N434Y / Y436VF10376.10E−08L235K / S239K / M252Y / T307Q / Q311A / M428I / N434Y / Y436VF10382.80E−07L235K / P238D / S239K / M252Y / N434YF10391.30E−07L235K / P238D / S239K / M252Y / N434Y / Y436VF10402.00E−07L235K / S239K / T250V / M252Y / N434Y / Y436VF10411.40E−08L235K / S239K / T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF10422.00E−07L235K / S239K / M252Y / T307A / Q311H / N434YF10435.20E−08L235K / S239K / T250V / M252Y / V308P / N434YF10443.50E−08L235K / S239K / T250V / M252Y / T307Q / V308P / Q311A / N434YF10452.50E−08L235K / S239K / M252Y / T307Q / V308P / Q311A / N434YF10464.50E−09L235K / S239K / T250V / M252Y / N286E / T307Q / V308P / Q311A / N434Y / Y436VF10473.40E−09L235K / S239K / M252Y / N286E / T307Q / V308P / Q311A / N434Y / Y436VF10489.90E−08L235K / S239K / M252Y / T307A / Q311H / N434Y / Y436VF10503.50E−09T250V / M252Y / N286E / T307Q / V308P / Q311A / M428I / N434Y / Y436VF10513.90E−09L235R / S239K / T250V / M252Y / N286E / T307Q / V308P / Q311A / M428I / N434Y / Y436VF10523.20E−09L235R / S239K / M252Y / N286E / T307Q / V308P / Q311A / M428I / N434Y / Y436V

[0838] TABLE 5-26F10534.23E−08L235R / S239K / T250V / M252Y / T307Q / Q311A / N434Y / Y436VF10581.31E−07M252Y / Q386E / N434Y / Y436VF10591.39E−07M252Y / Q386R / N434Y / Y436VF10601.43E−07M252Y / Q386S / N434Y / Y436VF10611.19E−07M252Y / P387E / N434Y / Y436VF10621.20E−07M252Y / P387R / N434Y / Y436VF10631.43E−07M252Y / P387S / N434Y / Y436VF10641.32E−07M252Y / V422E / N434Y / Y436VF10651.38E−07M252Y / V422R / N434Y / Y436VF10661.45E−07M252Y / V422S / N434Y / Y436VF10671.26E−07M252Y / S424E / N434Y / Y436VF10681.69E−07M252Y / S424R / N434Y / Y436VF10691.39E−07M252Y / N434Y / Y436V / Q438EF10701.73E−07M252Y / N434Y / Y436V / Q438RF10711.24E−07M252Y / N434Y / Y436V / Q438SF10721.35E−07M252Y / N434Y / Y436V / S440EF10731.34E−07M252Y / N434Y / Y436V / S440RF10741.32E−07S239D / M252Y / N434Y / Y436VF10751.40E−07M252Y / K326D / L328Y / N434Y / Y436VF10761.27E−07S239D / M252Y / K326D / L328Y / N434Y / Y436VF10772.03E−06K248N / M252Y / N434YF10784.70E−07M252Y / E380N / E382S / N434YF10793.44E−07M252Y / E382N / N384S / N434YF10803.19E−07M252Y / S424N / N434YF10816.20E−07M252Y / N434Y / Y436N / Q438TF10822.76E−07M252Y / N434Y / Q438NF10833.45E−07M252Y / N434Y / S440NF10942.60E−07M252Y / N434Y / S442NF10952.86E−07M252Y / S383N / G385S / N434YF10962.72E−07M252Y / Q386T / N434YF10972.82E−07M252Y / G385N / P387S / N434YF10982.58E−07S239D / M252Y / N434YF10992.57E−07M252Y / K326D / L328Y / N434YF11002.41E−07S239D / M252Y / K326D / L328Y / N434YF11016.59E−08S239D / M252Y / T307Q / Q311A / N434YF11026.46E−08M252Y / T307Q / Q311A / K326D / L328Y / N434YF11036.11E−08S239D / M252Y / T307Q / Q311A / K326D / L328Y / N434YF11041.77E−07M252Y / V422E / S424R / N434Y / Y436VF11051.54E−07M252Y / V422S / S424R / N434Y / Y436VF11061.42E−07M252Y / N434Y / Y436V / Q438R / S440EF11071.23E−07M252Y / V422D / N434Y / Y436VF11081.26E−07M252Y / V422K / N434Y / Y436VF11091.27E−07M252Y / V422T / N434Y / Y436VF11101.33E−07M252Y / V422Q / N434Y / Y436V

[0839] TABLE 5-27F11111.65E−07M252Y / S424K / N434Y / Y436VF11121.23E−07M252Y / N434Y / Y436V / Q438KF11131.18E−07M252Y / N434Y / Y436V / S440DF11141.31E−07M252Y / N434Y / Y436V / S440QF11151.35E−07M252Y / S424N / N434Y / Y436VF11167.44E−08M252Y / T307Q / Q311A / S424N / N434YF11174.87E−08T250V / M252Y / T307Q / Q311A / S424N / N434Y / Y436VF11181.32E−08T250V / M252Y / T307Q / V308P / Q311A / S424N / N434Y / Y436VF11191.03E−08T250V / M252Y / T307Q / V308P / Q311A / V422E / N434Y / Y436VF11201.04E−08T250V / M252Y / T307Q / V308P / Q311A / S424R / N434Y / Y436VF11211.04E−08T250V / M252Y / T307Q / V308P / Q311A / V422E / S424R / N434Y / Y436VF11221.37E−08T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436V / Q438RF11239.55E−09T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436V / S440EF11241.22E−08T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436V / Q438R / S440EF11255.18E−08M252Y / T307Q / N434Y / Y436VF11268.95E−08M252Y / T307A / N434Y / Y436VF11277.94E−08M252Y / Q311A / N434Y / Y436VF11281.17E−07M252Y / Q311H / N434Y / Y436VF11294.48E−08M252Y / T307Q / Q311H / N434Y / Y436VF11305.54E−08M252Y / T307A / Q311A / N434Y / Y436VF11311.29E−07L235R / S239K / M252Y / V422E / N434Y / Y436VF11321.40E−07L235R / S239K / M252Y / V422S / N434Y / Y436VF11331.58E−07L235R / S239K / M252Y / S424R / N434Y / Y436VF11341.66E−07L235R / S239K / M252Y / N434Y / Y436V / Q438RF11351.26E−07L235R / S239K / M252Y / N434Y / Y436V / S440EF11361.63E−07L235R / S239K / M252Y / V422E / S424R / N434Y / Y436VF11371.58E−07L235R / S239K / M252Y / V422S / S424R / N434Y / Y436VF11381.65E−07L235R / S239K / M252Y / N434Y / Y436V / Q438R / S440EF11391.52E−07L235R / S239K / M252Y / S424N / N434Y / Y436VF11401.62E−07M252Y / V422E / S424R / N434Y / Y436V / Q438R / S440EF11411.77E−07M252Y / V422S / S424R / N434Y / Y436V / Q438R / S440EF11421.87E−07L235R / S239K / M252Y / V422E / S424R / N434Y / Y436V / Q438R / S440EF11431.98E−07L235R / S239K / M252Y / V422S / S424R / N434Y / Y436V / Q438R / S440EF11441.44E−08L235R / S239K / T250V / M252Y / T307Q / V308P / Q311A / N434Y / Y436V / Q438R / S440EF11455.23E−08T250V / M252Y / T307Q / Q311A / N434Y / Y436V / Q438R / S440EF11466.24E−08L235R / S239K / T250V / M252Y / T307Q / Q311A / N434Y / Y436V / Q438R / S440EF11477.19E−08M252Y / T307Q / Q311A / N434Y / Q438R / S440EF11487.63E−08L235R / S239K / M252Y / T307Q / Q311A / N434Y / Q438R / S440EF11512.51E−07L235R / S239K / M252Y / S424N / N434YF11527.38E−08L235R / S239K / M252Y / T307Q / Q311A / S424N / N434YF11534.85E−08L235R / S239K / T250V / M252Y / T307Q / Q311A / S424N / N434Y / Y436VF11541.34E−08L235R / S239K / T250V / M252Y / T307Q / V308P / Q311A / S424N / N434Y / Y436

[0840] TABLE 5-28VF11572.09E−07M252Y / N434Y / Q438R / S440EF11582.44E−07L235R / S239K / M252Y / N434Y / Q438R / S440EF11594.79E−07S424N / N434WF11602.88E−07V308F / S424N / N434YF11611.07E−06I332V / S424N / N434YF11623.43E−07P238D / T250Y / M252Y / N434Y / Y436VF11631.54E−07P238D / T250Y / M252Y / T307Q / Q311A / N434YF11646.96E−08P238D / T250Y / M252Y / T307Q / Q311A / N434Y / Y436VF11651.63E−08P238D / T250Y / M252Y / T307Q / V308P / Q311A / N434Y / Y436VF11744.90E−07P257I / N434HF11761.98E−06V308FF11788.72E−07V259I / V308F / M428LF11831.28E−06E380A / M428L / N434SF11841.00E−06T307A / M428L / N434SF11859.17E−07T307A / E380A / M428L / N434SF11881.72E−06T307A / E380A / N434HF11891.57E−07M252Y / H433D / N434Y / Y436V / Q438R / S440EF11902.40E−07M252Y / H433E / N434Y / Y436V / Q438R / S440EF11912.11E−07M252Y / N434Y / Y436V / T437A / Q438R / S440EF11921.27E−07M252Y / N434Y / Y436V / T437G / Q438R / S440EF11941.55E−07M252Y / N434Y / Y436V / Q438R / K439D / S440EF11951.76E−07M252Y / N434Y / Y436V / Q438R / S440E / L441AF11961.51E−07M252Y / N434Y / Y436V / Q438R / S440E / L441EF11979.46E−08M252Y / S254T / N434Y / Y436V / Q438R / S440EF11987.83E−08M252Y / T256E / N434Y / Y436V / Q438R / S440EF11996.25E−08M252Y / S254T / T256E / N434Y / Y436V / Q438R / S440EF12001.26E−07T250V / M252Y / S254T / N434Y / Y436V / Q438R / S440EF12011.07E−07T250V / M252Y / T256E / N434Y / Y436V / Q438R / S440EF12028.81E−08T250V / M252Y / S254T / T256E / N434Y / Y436V / Q438R / S440EF12031.52E−07M252Y / T256Q / N434Y / Y436V / Q438R / S440EF12041.18E−07M252Y / S254T / T256Q / N434Y / Y436V / Q438R / S440EF12051.98E−07T250V / M252Y / T256Q / N434Y / Y436V / Q438R / S440EF12061.69E−07T250V / M252Y / S254T / T256Q / N434Y / Y436V / Q438R / S440EF12071.11E−06I332E / M428L / N434SF12085.71E−07L251A / M252Y / N434Y / Y436VF12111.23E−06L251H / M252Y / N434Y / Y436VF12136.33E−07L251N / M252Y / N434Y / Y436VF12161.16E−06L251S / M252Y / N434Y / Y436VF12171.14E−06L251T / M252Y / N434Y / Y436VF12182.51E−07L251V / M252Y / N434Y / Y436VF12292.81E−06M252Y / I253V / N434Y / Y436VF12301.12E−07M252Y / N434Y / Y436V / Q438R / S440DF12319.73E−08M252Y / N434Y / Y436V / Q438K / S440E

[0841] TABLE 5-29F12329.79E−08M252Y / N434Y / Y436V / Q438K / S440DF12431.25E−07L235R / S239K / M252Y / S254T / N434Y / Y436V / Q438R / S440EF12441.02E−07L235R / S239K / M252Y / T256E / N434Y / Y436V / Q438R / S440EF12458.20E−08L235R / S239K / M252Y / S254T / T256E / N434Y / Y436V / Q438R / S4...

Claims

1. A method of producing a pharmaceutical composition that induces humoral immunity to a target antigen, the method comprising:identifying the amino acid sequence of a first antigen-binding molecule comprising(i) an antigen-binding domain comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the antigen-binding domain binds to the target antigen with an antigen-binding activity that is higher at pH 7.4 than at pH 5.8, and the VL comprises a histidine at one or more of Kabat numbering positions 24, 27, 28, 31, 32, 34, 50, 51, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, 94, and 95A, and(ii) a first FcRn-binding domain;generating a DNA encoding a second antigen-binding molecule comprising the antigen-binding domain and a non-native human IgG1 Fc region comprising a second FcRn-binding domain, wherein the amino acid sequence of the second FcRn-binding domain differs from the amino acid sequence of the first FcRn-binding domain by substitution at one or more positions, wherein at least one of the substitutions is a set of one or more substitutions that matches the set of substitutions present in any one of the variants F10, F12, F14, F32, F34, F36, F38, F40, F47, F49, F50, F53, F54, F58, F59, F61, F69, F71, F73, F78, F80, F82, F84, F86, F88, F90, F92, F93, F95, F101, F103 to F108, F111, F113 to F116 listed in Tables 5-1 to 5-3, wherein all position numbers in Tables 5-1 to 5-3 are according to EU numbering, and wherein the second FcRn-binding domain's binding to a human FcRn receptor at pH 7.4 is greater than the binding of the first FcRn-binding domain to a human FcRn receptor at the same pH;expressing the DNA, thereby producing the second antigen-binding molecule;collecting the second antigen-binding molecule; andproducing a pharmaceutical composition comprising the second antigen-binding molecule, wherein the pharmaceutical composition induces humoral immunity to the target antigen.

2. The method of claim 1, wherein the second antigen-binding molecule binds to and neutralizes the target antigen.

3. The method of claim 1, wherein the second antigen-binding molecule has cytotoxic activity against a cell expressing the target antigen.

4. The method of claim 1, wherein the non-native human IgG1 Fc region differs from a native human IgG1 Fc region at one or more positions, including one or more of the following EU numbering positions: 239, 252, 257, 286, 307, 308, 428, and 434.

5. The method of claim 1, wherein one or more of the following EU numbering positions in the non-native human IgG1 Fc region is occupied by the indicated amino acid:Ala at position 257;Pro at position 308;Leu at position 428;Tyr at position 434.

6. The method of claim 1, wherein the non-native human IgG1 Fc region's binding to a human Fcγ receptor is greater than a native human IgG1 Fc region's binding to the human Fcγ receptor, wherein the native human IgG1 Fc region comprises a fucose-containing sugar chain bound at EU numbering position 297.

7. The method of claim 6, wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

8. The method of claim 1, wherein the non-native human IgG1 Fc region differs from a native human IgG1 Fc region at one or more positions, including one or more of the following EU numbering positions: 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, 440.

9. The method of claim 6, wherein one or more of the following EU numbering positions in the non-native human IgG1 Fc region is occupied by the indicated amino acid:either Lys or Tyr at position 221;any one of Phe, Trp, Glu, and Tyr at position 222;any one of Phe, Trp, Glu, and Lys at position 223;any one of Phe, Trp, Glu, and Tyr at position 224;any one of Glu, Lys, and Trp at position 225;any one of Glu, Gly, Lys, and Tyr at position 227;any one of Glu, Gly, Lys, and Tyr at position 228;any one of Ala, Glu, Gly, and Tyr at position 230;any one of Glu, Gly, Lys, Pro, and Tyr at position 231;any one of Glu, Gly, Lys, and Tyr at position 232;any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 233;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 234;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 235;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 236;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 237;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 238;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 239;any one of Ala, Ile, Met, and Thr at position 240;any one of Asp, Glu, Leu, Arg, Trp, and Tyr at position 241;any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at position 243;His at position 244;Ala at position 245;any one of Asp, Glu, His, and Tyr at position 246;any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at position 247;any one of Glu, His, Gln, and Tyr at position 249;either Glu or Gln at position 250;Phe at position 251;any one of Phe, Met, and Tyr at position 254;any one of Glu, Leu, and Tyr at position 255;any one of Ala, Met, and Pro at position 256;any one of Asp, Glu, His, Ser, and Tyr at position 258;any one of Asp, Glu, His, and Tyr at position 260;any one of Ala, Glu, Phe, Ile, and Thr at position 262;any one of Ala, Ile, Met, and Thr at position 263;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 264;any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr at position 265;any one of Ala, Ile, Met, and Thr at position 266;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 267;any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at position 268;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 269;any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 270;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 271;any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 272;either Phe or Ile at position 273;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 274;either Leu or Trp at position 275;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 276;any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 278;Ala at position 279;any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at position 280;any one of Asp, Lys, Pro, and Tyr at position 281;any one of Glu, Gly, Lys, Pro, and Tyr at position 282;any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at position 283;any one of Asp, Glu, Leu, Asn, Thr, and Tyr at position 284;any one of Asp, Glu, Lys, Gln, Trp, and Tyr at position 285;any one of Glu, Gly, Pro, and Tyr at position 286;any one of Asn, Asp, Glu, and Tyr at position 288;any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at position 290;any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at position 291;any one of Ala, Asp, Glu, Pro, Thr, and Tyr at position 292;any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 293;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 294;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 295;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at position 296;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 297;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at position 298;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at position 299;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 300;any one of Asp, Glu, His, and Tyr at position 301;Ile at position 302;any one of Asp, Gly, and Tyr at position 303;any one of Asp, His, Leu, Asn, and Thr at position 304;any one of Glu, Ile, Thr, and Tyr at position 305;any one of Ala, Asp, Asn, Thr, Val, and Tyr at position 311;Phe at position 313;Leu at position 315;either Glu or Gln at position 317;any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at position 318;any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at position 320;any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at position 322;Ile at position 323;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at position 324;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 325;any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at position 326;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at position 327;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 328;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 329;any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 330;any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at position 331;any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 332;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at position 333;any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at position 334;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at position 335;any one of Glu, Lys, and Tyr at position 336;any one of Glu, His, and Asn at position 337;any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at position 339;either Ala or Val at position 376;either Gly or Lys at position 377;Asp at position 378;Asn at position 379;any one of Ala, Asn, and Ser at position 380;either Ala or Ile at position 382;Glu at position 385;Thr at position 392;Leu at position 396;Lys at position 421;Asn at position 427;either Phe or Leu at position 428;Met at position 429;Trp at position 434;Ile at position 436;any one of Gly, His, Ile, Leu, and Tyr at position 440.

10. The method of claim 6, wherein the percentage of the second antigen-binding molecule that has a fucose-deficient sugar chain bound at EU numbering position 297 in the pharmaceutical composition is higher than the percentage of a naturally-occurring IgG1 that has a fucose-deficient sugar chain bound at EU numbering position 297 in a composition of the naturally-occurring IgG1.

11. The method of claim 6, wherein the percentage of the second antigen-binding molecule that has a bisecting N-acetylglucosamine sugar chain bound at EU numbering position 297 in the pharmaceutical composition is higher than the percentage of a naturally-occurring IgG1 that has a bisecting N-acetylglucosamine sugar chain bound at EU numbering position 297 in a composition of the naturally-occurring IgG.

12. A method of producing a pharmaceutical composition that induces humoral immunity to a target antigen, the method comprising:(a) identifying the amino acid sequence of a first antigen-binding molecule comprising a first FcRn-binding domain and an antigen-binding domain comprising a VH and a VL, wherein the antigen-binding domain binds to the target antigen, and the VL comprises a histidine at one or more of Kabat numbering positions 24, 27, 28, 31, 32, 34, 50, 51, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, 94, and 95A;(b) testing the antigen-binding domain's antigen-binding activity at a first pH between pH 6.7-10.0 and at a second pH between pH 4.0-6.5, and determining that the antigen-binding activity is greater at the first pH than at the second pH;(c) generating a DNA encoding a second antigen-binding molecule comprising the antigen-binding domain and a non-native human IgG1 Fc region comprising a second FcRn-binding domain that binds to a human FcRn receptor at pH 7.4, wherein the amino acid sequence of the second FcRn-binding domain differs from the amino acid sequence of the first FcRn-binding domain by substitution at one or more positions, wherein at least one of the substitutions is a set of one or more substitutions that matches the set of substitutions present in any one of variants F10, F12, F14, F25, F32, F34, F36, F38, F40, F47, F49, F50, F53, F54, F58, F59, F61, F69, F71, F73, F78, F80, F82, F84, F86, F88, F90, F92, F93, F95, F101, F103 to F108, F111, F113 to F116 listed in Tables 5-1 to 5-3; wherein all position numbers in Tables 5-1 to 5-3 are according to EU numbering,(d) culturing cells comprising the DNA, so that the cells express the second antigen-binding molecule;(e) collecting the second antigen-binding molecule; and(f) producing a pharmaceutical composition comprising the second antigen-binding molecule, wherein the pharmaceutical composition induces humoral immunity to the target antigen.

13. The method of claim 12, wherein the second antigen-binding molecule is an antibody.

14. The method of claim 12, wherein the second antigen-binding molecule binds to and neutralizes the target antigen.

15. The method of claim 12, wherein the second antigen-binding molecule has cytotoxic activity against a cell expressing the target antigen.

16. The method of claim 12, wherein the non-native human IgG1 Fc region differs from a native human IgG Fc region at one or more of the following EU numbering positions: 257, 308, 428, 434.

17. The method of claim 12, wherein one or more of the following EU numbering positions in the non-native human IgG1 Fc region is occupied by the indicated amino acid:Ala at position 257;Pro at position 308;Leu at position 428;Tyr at position 434.

18. The method of claim 12, wherein the non-native human IgG1 Fc region's binding to a human Fcγ receptor is greater than a native human IgG Fc region's binding to the human Fcγ receptor, wherein the native human IgG Fc region comprises a fucose-containing sugar chain bound at EU numbering position 297.

19. The method of claim 18, wherein the human Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).

20. The method of claim 18, wherein one or more of the following EU numbering positions in the non-native human IgG1 Fc region is occupied by the indicated amino acid:either Lys or Tyr at position 221;any one of Phe, Trp, Glu, and Tyr at position 222;any one of Phe, Trp, Glu, and Lys at position 223;any one of Phe, Trp, Glu, and Tyr at position 224;any one of Glu, Lys, and Trp at position 225;any one of Glu, Gly, Lys, and Tyr at position 227;any one of Glu, Gly, Lys, and Tyr at position 228;any one of Ala, Glu, Gly, and Tyr at position 230;any one of Glu, Gly, Lys, Pro, and Tyr at position 231;any one of Glu, Gly, Lys, and Tyr at position 232;any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 233;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 234;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 235;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 236;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 237;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 238;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 239;any one of Ala, Ile, Met, and Thr at position 240;any one of Asp, Glu, Leu, Arg, Trp, and Tyr at position 241;any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at position 243;His at position 244;Ala at position 245;any one of Asp, Glu, His, and Tyr at position 246;any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at position 247;any one of Glu, His, Gln, and Tyr at position 249;either Glu or Gln at position 250;Phe at position 251;any one of Phe, Met, and Tyr at position 254;any one of Glu, Leu, and Tyr at position 255;any one of Ala, Met, and Pro at position 256;any one of Asp, Glu, His, Ser, and Tyr at position 258;any one of Asp, Glu, His, and Tyr at position 260;any one of Ala, Glu, Phe, Ile, and Thr at position 262;any one of Ala, Ile, Met, and Thr at position 263;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 264;any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr at position 265;any one of Ala, Ile, Met, and Thr at position 266;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 267;any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at position 268;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 269;any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 270;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 271;any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 272;either Phe or Ile at position 273;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 274;either Leu or Trp at position 275;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 276;any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 278;Ala at position 279;any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at position 280;any one of Asp, Lys, Pro, and Tyr at position 281;any one of Glu, Gly, Lys, Pro, and Tyr at position 282;any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at position 283;any one of Asp, Glu, Leu, Asn, Thr, and Tyr at position 284;any one of Asp, Glu, Lys, Gln, Trp, and Tyr at position 285;any one of Glu, Gly, Pro, and Tyr at position 286;any one of Asn, Asp, Glu, and Tyr at position 288;any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at position 290;any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at position 291;any one of Ala, Asp, Glu, Pro, Thr, and Tyr at position 292;any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 293;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 294;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 295;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at position 296;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 297;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at position 298;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at position 299;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 300;any one of Asp, Glu, His, and Tyr at position 301;Ile at position 302;any one of Asp, Gly, and Tyr at position 303;any one of Asp, His, Leu, Asn, and Thr at position 304;any one of Glu, Ile, Thr, and Tyr at position 305;any one of Ala, Asp, Asn, Thr, Val, and Tyr at position 311;Phe at position 313;Leu at position 315;either Glu or Gln at position 317;any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at position 318;any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at position 320;any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at position 322;Ile at position 323;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at position 324;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 325;any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at position 326;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at position 327;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 328;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 329;any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 330;any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at position 331;any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 332;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at position 333;any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at position 334;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at position 335;any one of Glu, Lys, and Tyr at position 336;any one of Glu, His, and Asn at position 337;any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at position 339;either Ala or Val at position 376;either Gly or Lys at position 377;Asp at position 378;Asn at position 379;any one of Ala, Asn, and Ser at position 380;either Ala or Ile at position 382;Glu at position 385;Thr at position 392;Leu at position 396;Lys at position 421;Asn at position 427;either Phe or Leu at position 428;Met at position 429;Trp at position 434;Ile at position 436;any one of Gly, His, Ile, Leu, and Tyr at position 440.

21. The method of claim 18, wherein the percentage of the second antigen-binding molecule that has a fucose-deficient sugar chain bound at EU numbering position 297 in the pharmaceutical composition is higher than the percentage of a naturally-occurring IgG that has a fucose-deficient sugar chain bound at EU numbering position 297 in a composition of the naturally-occurring IgG.

22. The method of claim 18, wherein the percentage of the second antigen-binding molecule that has a bisecting N-acetylglucosamine sugar chain bound at EU numbering position 297 in the pharmaceutical composition is higher than the percentage of a naturally-occurring IgG that has a bisecting N-acetylglucosamine sugar chain bound at EU numbering position 297 in a composition of the naturally-occurring IgG.

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